U.S. patent application number 16/174938 was filed with the patent office on 2019-06-06 for aryl imidazoles for treatment of cancer.
The applicant listed for this patent is Aptose Biosciences Inc.. Invention is credited to Stephen H. HOWELL, William G. RICE, Cheng-Yu TSAI.
Application Number | 20190169215 16/174938 |
Document ID | / |
Family ID | 66333658 |
Filed Date | 2019-06-06 |
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United States Patent
Application |
20190169215 |
Kind Code |
A1 |
RICE; William G. ; et
al. |
June 6, 2019 |
ARYL IMIDAZOLES FOR TREATMENT OF CANCER
Abstract
The present invention relates to a method of preventing,
reducing, or treating cancer in a subject, comprising administering
a therapeutically effective amount of ##STR00001## or a
pharmaceutically acceptable salt, free base, hydrate, complex, or
chelate (including metal chelates, such as iron, zinc and others)
thereof to the subject, wherein the subject has a mutation in a DNA
repair gene.
Inventors: |
RICE; William G.; (Del Mar,
CA) ; HOWELL; Stephen H.; (La Jolla, CA) ;
TSAI; Cheng-Yu; (La Jolla, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Aptose Biosciences Inc. |
Mississauga |
|
CA |
|
|
Family ID: |
66333658 |
Appl. No.: |
16/174938 |
Filed: |
October 30, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62578938 |
Oct 30, 2017 |
|
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|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 31/4745 20130101; C07D 471/14 20130101; A61K 45/06 20130101;
C07F 15/025 20130101; A61K 31/555 20130101; A61K 31/517 20130101;
A61K 31/4745 20130101; A61K 2300/00 20130101; A61K 31/555 20130101;
A61K 2300/00 20130101 |
International
Class: |
C07F 15/02 20060101
C07F015/02; A61P 35/00 20060101 A61P035/00; A61K 31/517 20060101
A61K031/517; C07D 471/14 20060101 C07D471/14 |
Claims
1. A method of preventing, reducing or treating cancer in a
subject, comprising administering a therapeutically effective
amount of Compound I: ##STR00013## or a pharmaceutically acceptable
salt, free base, hydrate, complex, or chelate thereof to the
subject; wherein the subject has a mutation in a DNA repair
gene.
2. The method of claim 1, wherein the DNA repair gene is a
homologous recombinant gene.
3. The method of claim 1, wherein the DNA repair gene is a gene in
the homologous recombination (HR) dependent deoxyribonucleic acid
(DNA) double strand break (DSB) repair pathway.
4. The method of claim 1, wherein the DNA repair gene is one or
more genes selected from the group consisting of BRCA-1, BRCA-2,
ATM, ATR, CHK1, CHK2, Rad51, RPA and XRCC3.
5. The method of claim 2, wherein the DNA repair gene is BRCA-1
and/or BRCA-2.
6. The method of claim 1, wherein the subject is heterozygous for a
mutation in a DNA repair gene.
7. The method of claim 6, wherein the subject is heterozygous for a
mutation in a gene in the homologous recombination (HR) dependent
deoxyribonucleic acid (DNA) double strand break (DSB) repair
pathway.
8. The method of claim 6, wherein the subject is heterozygous for a
mutation in BRCA1 or BRCA2.
9. The method of claim 6, wherein the subject is homozygous for a
mutation in BRCA1 or BRCA2.
10. The method of claim 1, wherein the cancer is selected from the
group consisting of heme cancer, colorectal cancer, ovarian cancer,
breast cancer, cervical cancer, lung cancer, liver cancer,
pancreatic cancer, cancer of the lymph nodes, leukemia, renal
cancer, colon cancer, prostate cancer, brain cancer, cancer of the
head and neck, bone cancer, carcinoma of the larynx and oral
cavity, Ewing's sarcoma, skin cancer, kidney cancer, and cancer of
the heart.
11. The method of claim 10, wherein the cancer is selected from the
group consisting of breast cancer, lung cancer, cancer of the lymph
nodes, colon cancer, leukemia, renal cancer, and prostate
cancer.
12. The method of claim 11, wherein the cancer is breast
cancer.
13. The method of claim 1, wherein the cancer is a BRCA-associated
cancer.
14. The method of claim 13, wherein the BRCA-associated cancer has
one or more mutations of the BRCA-1 and/or BRCA-2 genes.
15. The method of claim 1, wherein the subject is human.
16. The method of claim 1, further comprising the administering of
a therapeutically effective amount of a second therapeutically
active agent.
17. The method of claim 16, wherein the second therapeutically
active agent is administered before, during, or after the subject
has been administered Compound I.
18. The method of claim 16, wherein the second therapeutically
active agent is selected from one or more of the group consisting
of immunotherapeutic agents, anticancer agents, and angiogenic
agents.
19. The method of claim 18, wherein the second therapeutically
active agent is a PARP inhibitor.
20. The method of claim 19, wherein the PARP inhibitor is
olaparib.
21. The method of claim 1, wherein the subject experiences less
than a 90% decrease in bone marrow activity relative to a subject
who was not administered a therapeutically effective amount of
Compound I ##STR00014## or a pharmaceutically acceptable salt, free
base, hydrate, complex, or chelate thereof.
22. The method of claim 21, wherein the subject experiences less
than a 10% decrease in bone marrow activity.
23. The method of claim 21, wherein the subject experiences no
decrease in bone marrow activity.
24. The method of claim 1, wherein the subject already has
cancer.
25. The method of claim 24, wherein the subject experiences a
reduction or decrease in size of a tumor associated with a
cancer.
26. The method of claim 25, wherein the subject experiences
complete elimination of the tumor associated with cancer.
27. The method of claim 24, wherein the subject experiences an
inhibition, decrease, or reduction of neo-vascularization or
angiogenesis in a tumor associated with a cancer.
28. A method for killing cancer cells, comprising contacting said
cells with a therapeutically effective amount of Compound I
##STR00015## or a pharmaceutically acceptable salt, free base,
hydrate, complex, or chelate thereof.
29. The method of claim 28, wherein the cancer cells have a
deficiency in one or more genes selected from the group consisting
of BRCA-1, BRCA-2, ATM, ATR, CHK1, CHK2, Rad51, RPA and XRCC3.
30. A method for inducing cell cycle arrest in cancer cells,
comprising contacting said cells with a therapeutically effective
amount of Compound I ##STR00016## or a pharmaceutically acceptable
salt, free base, hydrate, complex, or chelate thereof.
31. The method of claim 30, wherein the cancer cells have a
deficiency in one or more genes selected from the group consisting
of BRCA-1, BRCA-2, ATM, ATR, CHK1, CHK2, Rad51, RPA and XRCC3.
32. A method of preventing, reducing or treating cancer in a
subject, comprising administering a therapeutically effective
amount of one or more molecules of ##STR00017## in complex with one
or more metal atoms, wherein the subject has a mutation in a DNA
repair gene.
33. The method of claim 32, wherein the one or more metal atoms are
selected from the group consisting of iron, zinc, aluminum,
magnesium, platinum, silver, gold, chromium, nickel, titanium,
copper, scandium, zirconium, vanadium, molybdenum, manganese,
tungsten and cobalt.
34. The method of claim 33, wherein the one or more metal atoms are
iron.
35. The method of claim 34, wherein the complex has the following
structure: ##STR00018##
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/578,938, filed on Oct. 30, 2017, the contents of
which is hereby incorporated by reference in its entirety.
DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY
[0002] The contents of the text file submitted electronically
herewith are incorporated herein by reference in their entirety: A
computer readable format copy of the Sequence Listing (filename:
LOTH_057_01US_SeqList_ST25, date recorded: Nov. 29, 2018, file size
.about.4.63 kilobytes).
FIELD OF THE INVENTION
[0003] The present invention generally relates to a method of
preventing, reducing, or treating cancer in a subject.
BACKGROUND
[0004] Proteins encoded by the breast cancer susceptibility genes
(BRCA proteins) have been associated with a predisposition to
breast, ovarian and other cancers. These proteins are ubiquitously
expressed thereby implicating them in many processes fundamental to
all cells including DNA repair and recombination, checkpoint
control of cell cycle and transcription.
[0005] Specifically, genetic susceptibility to breast cancer has
been linked to mutations of certain genes (e.g., BRCA-1 and
BRCA-2). Proteins encoded by these genes are believed to work to
preserve chromosome structure, but their precise role is unclear
due to them being involved in a multitude of processes. It is
postulated that a mutation causes a disruption in the protein which
causes chromosomal instability in BRCA deficient cells thereby
predisposing them to neoplastic transformation.
[0006] About 10% of breast cancer cases cluster in families, some
due to mutations in the BRCA-1 and BRCA-2 genes, giving rise to
higher cancer risk. Mutations in other genes linked to tumor
suppression may account for cancer predisposition. These include
mutations in p53 tumor suppression, the STK11/LKB, protein kinase
or the PTEN phosphatase.
[0007] Deficits in homologous recombination in tumors provide the
opportunity for selective killing of tumor cells; however, the
drugs currently used to exploit this opportunity cause serious
myelosuppression which limits dose. Therefore, there is still an
unmet need of high priority in the art to identify drugs for which
loss of BRCA1 or BRCA2 function results in hypersensitivity but
that do not cause myelosuppression.
SUMMARY OF THE INVENTION
[0008] The present disclosure is related to a method of preventing,
reducing, or treating cancer in a subject.
[0009] In an embodiment, the present disclosure relates to a method
of preventing, reducing, or treating cancer in a subject,
comprising administering a therapeutically effective amount of
compound I,
##STR00002##
or a pharmaceutically acceptable salt, free base, hydrate, complex,
or chelate (including metal chelates, such as iron, zinc and
others) thereof to the subject, wherein the subject has a mutation
in a DNA repair gene. In certain embodiments, the DNA repair gene
is a homologous recombinant gene. For example, the DNA repair gene
is a gene in the homologous recombination (HR) dependent
deoxyribonucleic acid (DNA) double strand break (DSB) repair
pathway. In some embodiments, the DNA repair gene is one or more
genes selected from the group consisting of BRCA-1, BRCA-2, ATM,
ATR, CHK1, CHK2, Rad51, RPA and XRCC3. For example, the DNA repair
gene is BRCA-1 and/or BRCA-2. In an embodiment, the subject is
human.
[0010] In an embodiment, the subject is heterozygous for a mutation
in a DNA repair gene. In certain embodiments, the subject is
heterozygous for a mutation in a gene in the homologous
recombination (HR) dependent deoxyribonucleic acid (DNA) double
strand break (DSB) repair pathway. In one embodiment, the subject
is heterozygous for a mutation in BRCA1 or BRCA2. In another
embodiment, the subject is homozygous for a mutation in BRCA1 or
BRCA2.
[0011] In an embodiment, the cancer is selected from the group
consisting of a hematologic cancer, colorectal cancer, ovarian
cancer, breast cancer, cervical cancer, lung cancer, liver cancer,
pancreatic cancer, cancer of the lymph nodes, leukemia, renal
cancer, colon cancer, prostate cancer, brain cancer, cancer of the
head and neck, bone cancer, carcinoma of the larynx and oral
cavity, Ewing's sarcoma, skin cancer, kidney cancer, and cancer of
the heart. In certain embodiments, the cancer is selected from the
group consisting of breast cancer, lung cancer, ovarian cancer,
cancer of the lymph nodes, colon cancer, leukemia, renal cancer,
and prostate cancer. In one embodiment, the cancer is breast
cancer.
[0012] In some embodiments, the cancer is a hematological
malignancy. Examples of hematological malignancies include, but are
not limited to, leukemias, lymphomas, Hodgkin's disease, and
myeloma. Also, acute lymphocytic leukemia (ALL), acute myeloid
leukemia (AML), acute promyelocytic leukemia (APL), chronic
lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic
neutrophilic leukemia (CNL), acute undifferentiated leukemia (AUL),
anaplastic large-cell lymphoma (ALCL), prolymphocytic leukemia
(PML), juvenile myelomonocytic leukemia (JMML), adult T-cell ALL,
AML, with trilineage myelodysplasia (AMLITMDS), mixed lineage
leukemia (MLL), eosinophilic leukemia, mantle cell lymphoma,
myelodysplastic syndromes (MDSs) (e.g. high-risk MDS),
myeloproliferative disorders (MPD), and multiple myeloma (MM). In
some embodiments, the cancer is acute myeloid leukemia. In some
embodiments, the cancer is chronic myeloid leukemia. In some
embodiments, the cancer is a lymphoma. In some embodiments, the
cancer is high-risk myelodysplastic syndrome.
[0013] In an embodiment, the cancer is a BRCA-associated cancer. In
certain embodiments, the BRCA-associated cancer has one or more
mutations of the BRCA-1 and/or BRCA-2 genes.
[0014] In an embodiment, the method of the present disclosure
further comprises the administering of a therapeutically effective
amount of a second therapeutically active agent. The second
therapeutically active agent is administered before, during, or
after the subject has been administered
##STR00003##
The second therapeutically active agent is selected from one or
more of the group consisting of immunotherapeutic agents,
anticancer agents, and angiogenic agents. In one embodiment, the
second therapeutically active agent is a PARP inhibitor. For
example, the PARP inhibitor is olaparib.
[0015] In an embodiment, the subject experiences less than a 90%
decrease in bone marrow activity relative to a subject who was not
administered a therapeutically effective amount of
##STR00004##
or a pharmaceutically acceptable salt, free base, hydrate, complex,
or chelate (including metal chelates, such as iron, zinc and
others) thereof. For example, the subject may experience less than
a 10% decrease in bone marrow activity or no decrease in bone
marrow activity.
[0016] In an embodiment, the subject already has cancer. In certain
embodiments, the subject already having cancer experiences a
reduction or decrease in size of a tumor associated with a cancer.
For example, the subject experiences complete elimination of the
tumor associated with cancer. In certain embodiments, the subject
already having cancer experiences an inhibition, decrease, or
reduction of neo-vascularization or angiogenesis in a tumor
associated with a cancer.
[0017] In another embodiment, the present disclosure relates to a
method for killing cancer cells, comprising contacting said cells
with a therapeutically effective amount of
##STR00005##
or a pharmaceutically acceptable salt, free base, hydrate, complex,
or chelate (including metal chelates, such as iron, zinc and
others) thereof. In one embodiment, the cancer cells have a
deficiency in one or more genes selected from the group consisting
of BRCA-1, BRCA-2, ATM, ATR, CHK1, CHK2, Rad51, RPA and XRCC3.
[0018] In another embodiment, the present disclosure relates to a
method for inducing cell cycle arrest in cancer cells, comprising
contacting said cells with a therapeutically effective amount of
cells thereby predisposing them to neoplastic transformation.
[0019] In another embodiment, the present disclosure relates to a
method of preventing, reducing or treating cancer in a subject,
comprising administering a therapeutically effective amount of one
or more molecules of
##STR00006##
in complex with one or more metal atoms, wherein the subject has a
mutation in a DNA repair gene. In one embodiment, the one or more
metal atoms are selected from the group consisting of iron, zinc,
aluminum, magnesium, platinum, silver, gold, chromium, nickel,
titanium, copper, scandium, zirconium, vanadium, molybdenum,
manganese, tungsten and cobalt. In one embodiment, the one or more
metal atoms are iron. In certain embodiments, the complex has the
following structure:
##STR00007##
[0020] It should be appreciated that all combinations of the
foregoing concepts and additional concepts discussed in greater
detail below (provided such concepts are not mutually inconsistent)
are contemplated as being part of the inventive subject matter
disclosed herein. In particular, all combinations of claimed
subject matter appearing at the end of this disclosure are
contemplated as being part of the inventive subject matter
disclosed herein. It should also be appreciated that terminology
explicitly employed herein that also may appear in any disclosure
incorporated by reference should be accorded a meaning most
consistent with the particular concepts disclosed herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 shows that Fe(COMPOUND I).sub.3 is an active
intracellular form of COMPOUND I. (A) Structure of COMPOUND I. (B)
Structure of Fe(COMPOUND I).sub.3. (C) Relative cytotoxicity of
COMPOUND I (.box-solid.) and Fe(COMPOUND I).sub.3 (.circle-solid.)
in the Raji cells. (D) The intracellular accumulation of COMPOUND I
(.box-solid.) and Fe(COMPOUND I).sub.3 (.box-solid.) in Raji cells
exposed to 0.5 .mu.M COMPOUND I or Fe(COMPOUND I).sub.3 for 6 h.
Vertical bars, .+-.SEM; where missing SEM is less than the size of
the symbol; ***, P<0.001; ****, p<0.0001.
[0022] FIG. 2 shows that COMPOUND I causes DNA damage. (A) The
accumulation of phospho-ATM, .gamma.-H2AX and cleaved PARP in the
Raji cells as a function of duration of exposure to 0.5 .mu.M
COMPOUND I. The immunoblot shown is a representative of three
independent experiments. (B) Representative immunofluorescent
images of nuclear foci formation comparing DMSO- and COMPOUND
I-treated CAOV3 cells. (C) Mean .+-.SEM number of .gamma.H2AX foci
per cell; N=100. (D) Box and whisker plot showing neutral comment
assay quantification of percent tail DNA in CAOV3 cells treated
with DMSO or 0.5 .mu.M COMPOUND I for 6 h, N=number of cells
examined. Vertical bars, .+-.SEM; *, p<0.05, ****,
p<0.0001.
[0023] FIG. 3 shows that loss of BRCA1 and BRCA2 function results
in hypersensitivity to COMPOUND I. Sensitivity of BRCA1-proficient
and -deficient isogenic MCF10A clones (A), hTERT-IMEC clones (B)
and MCF7 (C) to olaparib (right) and COMPOUND I (left). Sensitivity
of BRCA2-proficient and -deficient isogenic PEO4 and PEO1 (D), and
HCT116 BRCA2-deficient clones (E) to olaparib and COMPOUND I. The
accumulation of g-H2AFX in the MCF7 control and shBRCA1 clone E7
cells (F) and the BRCA2-proficient HCT116 and the deficient clone
B18 cells treated with DMSO or the indicated concentration of
COMPOUND I for 24 hours (G). Vertical bars, .+-.SEM. *, P<0.05;
**, P<0.01; ***, P<0.001.
[0024] FIG. 4 shows characterization of cells resistant to COMPOUND
I (referred to as COMPOUND IR). (A) Concentration-survival curves
for Raji (.circle-solid.), Raji/COMPOUND IR (.box-solid.)
Raji/COMPOUND IR and Raji/COMPOUND IR cells after culture in
drug-free medium for 3 months (.tangle-solidup.). (B) Western blot
analysis of proteins involved in apoptosis in Raji and
Raji/COMPOUND IR treated with DMSO or COMPOUND I 0.5 .mu.M for 24
h. (C) The intracellular accumulation of COMPOUND I (.box-solid.)
and Fe(COMPOUND I).sub.3 (.box-solid.) in Raji and Raji/COMPOUND IR
cells after a 6 h exposure to 0.5 .mu.M COMPOUND I. (D) The
intracellular accumulation of Fe(COMPOUND I).sub.3 in the Raji and
Raji/COMPOUND IR cells at 6 h as a function of COMPOUND I
concentration. Vertical, bars, .+-.SEM; ** p<0.01; ***,
p<0.001; ****, p<0.0001.
[0025] FIG. 5 shows the role of ABCG2 in resistance to COMPOUND I.
(A) Relative levels of ABCG2 mRNA in the Raji and Raji/COMPOUND IR.
(B) Western blots of biotinylated proteins were probed with
anti-ABCG2 antibody. Na/K ATPase served as a loading control. (C)
Cytotoxicity of Ko143 in Raji (.circle-solid.) and Raji/COMPOUND IR
(.box-solid.). (D) Concentration-survival curves for Raji
(.circle-solid.) and Raji/COMPOUND IR (.box-solid.) treated with
COMPOUND I alone or in combination with COMPOUND I and 5 nM
(.tangle-solidup.) or 50 nM Ko143 (). (E) Cytotoxicity of topotecan
in Raji (.circle-solid.) and Raji/COMPOUND IR (.box-solid.) and the
combination of topotecan and 50 nM Ko143 in Raji/COMPOUND IR
(.tangle-solidup.). (F) Cytotoxicity of carboplatin in Raji
(.circle-solid.) and Raji/COMPOUND IR (.box-solid.) and the
combination of carboplatin and 50 nM Ko143 in Raji/COMPOUND IR
(.tangle-solidup.). (G) Concentration-survival curves for HEK-293
transfected with pcDNA (.circle-solid.) and ABCG2, clone R5
(.box-solid.) treated with COMPOUND I. Vertical, bars, .+-.SEM; **,
p<0.01.
[0026] FIG. 6 shows influx and efflux of COMPOUND I and Fe(COMPOUND
I).sub.3. (A) Time course of accumulation of COMPOUND I and
Fe(COMPOUND I).sub.3 into Raji and Raji/COMPOUND IR cells incubated
with 0.5 .mu.M COMPOUND I. (B) Time course of accumulation of
Fe(COMPOUND I).sub.3 into Raji and Raji/COMPOUND IR cells incubated
with 0.5 .mu.M Fe(COMPOUND I).sub.3. (C) Efflux of COMPOUND I and
Fe(COMPOUND I).sub.3 over 2 h from Raji and Raji/COMPOUND IR cells
loaded by exposure to 0.5 .mu.M COMPOUND I for 6 h.
[0027] FIG. 7 shows the accumulation of phospho-ATM, .gamma.-H2AX
and cleaved PARP in Raji compared to that in Raji/COMPOUND IR
cells.
[0028] FIG. 8 shows the antiproliferative activity of COMPOUND I
against leukemia and lymphoma cell lines. A) Concentration-response
curves for AML cell lines treated for 5 days with COMPOUND I. Cell
growth expressed as percent of growth of vehicle-treated cells. B)
Concentration-response curve for other leukemia and lymphoma cell
lines. Error bars, .+-.SD of at least three replicate assays.
[0029] FIG. 9 shows that COMPOUND I induces G0-G1 cell-cycle arrest
in a dose- and time-dependent manner in AML cell lines. A) Top,
MV4-11 cells treated with COMPOUND I at indicated concentrations
for 24 hours. Cell-cycle distribution assayed as described in the
Materials and Methods section. Bottom, CDK4 and CCND3 protein
levels in MV4-11 cells after 24-hour exposure to COMPOUND I.
Protein levels quantitated from three independent Western blots,
graphed as fold change over vehicle. B) and C) Effect of COMPOUND I
on cell-cycle distribution in KG-1 and EOL-1 cells. D)-F), Effect
of COMPOUND I on cell-cycle distribution as a function of duration
of exposure (MV4-11 cells,500 nmol/L; KG-1 cells, 600 nmol/L
COMPOUND I; and EOL-1 cells, 300 nmol/L COMPOUND I). Error bars,
.+-.SD of two replicate assays for flow cytometry and three
replicates for Western blots.
[0030] FIG. 10 shows that COMPOUND I treatment induces apoptosis in
a time- and concentration-dependent manner. A, Percent apoptotic
(early and late) MV4-11, KG-1, and EOL-1 cells after 24-hour
exposure to COMPOUND I. Apoptosis was measured as described in the
Materials and Methods section. B, Western blot analysis with
PARP1-specific antibody of AML cells treated for 24 hours with
V-vehicle or A-COMPOUND I. PARP1 antibody recognizes both
full-length (upper band) and cleaved PARP1 (lower band). C, Western
blot analysis of PARP1 cleavage in MV4-11, KG-1, and EOL-1 cells
treated for 1 to 24 hours with 500 nmol/L of COMPOUND I. GAPDH is
included as loading control. D, Time course of COMPOUND I induced
apoptosis in MV4-11 (500 nmol/L), KG-1 (600 nmol/L), and EOL-1 (300
nmol/L) cells. Error bars, .+-.SD of two replicate assays.
[0031] FIG. 11 shows that MYC RNA and protein expression is
negatively regulated by COMPOUND I. A, AML lines were treated for
24 hours and MYC mRNA levels measured by qRT-PCR with MYC-specific
primer/probe pairs. Graphed as percent of vehicle using GraphPad
Prism.b, Western blot analysis of MYC protein level in MV4-11,
KG-1, and EOL-1 cells treated for 24 hours at the concentrations
listed. GAPDH served as a loading control. C, Histogram plot of MYC
mRNA expression graphed as fold change over vehicle in MV4-11,
KG-1, and EOL-1 cells treated with 500 nmol/L COMPOUND I for the
times listed. D, Basal expression level of MYC mRNA in AML cell
lines compared with PBMCs from healthy donors. Expression relative
to GAPDH assayed by qRT-PCR. Error bars, .+-.SD from at least three
replicate experiments.
[0032] FIG. 12 shows that COMPOUND I induces DDR pathways. A, Total
TP53 protein levels in MV4-11 cells treated with (V) vehicle or 500
nmol/L (A) COMPOUND I for increasing periods of time. B,
Posttranslational modifications of TP53 detected by Western blot
analysis in MV4-11 cells treated as in A. C, Western blot analysis
of MV4-11 cells exposed to 500 nmol/L COMPOUND I. D, Western blot
analysis of .gamma.-H2AX (H2AX phos-S139) levels in MV4-11, KG-1,
and EOL-1 cells treated with COMPOUND I for 24 hours at the
concentrations noted.
[0033] FIG. 13 shows the in vitro and cellular activity of
Fe(COMPOUND I).sub.3 complex. A, Concentration-response curves for
AML cell lines treated for 5 days with parental COMPOUND I or
Fe(COMPOUND I).sub.3. Cell growth expressed as percent of growth of
vehicle-treated cells. Error bars, mean SD from 3-5 replicate
assays. B, Left, KLF4, CDKN1A, and MYC mRNA expression after
24-hour treatment with Fe(COMPOUND I).sub.3 at concentrations
listed. Error bars, mean .+-.SD. Right, Western blot analysis of
c-PARP1, MYC, and H2AX protein levels in MV4-11 cells after 24-hour
exposure to vehicle (v) or increasing concentrations of Fe(COMPOUND
I).sub.3. C, .DELTA.T.sub.1/2 values calculated from FRET curves
representative examples shown in FIGS. 22 and 23, with at least
three replicates for each curve. The .DELTA.T.sub.1/2 of each oligo
is plotted against log[drug] mol/L for each compound tested.
[0034] FIG. 14 shows the role of ABCG2 in resistance to Fe(COMPOUND
I).sub.3. (A) Concentration-survival cures for Raji
(.circle-solid.) and Raji/COMPOUND IR (.box-solid.) treated with
Fe(COMPOUND I).sub.3 alone or in combination with 50 nM Ko143 ().
(B) Concentration-survival curves for HEK-293 clone R5 transfected
with empty vector (.circle-solid.) or a vector expressing ABCG2
(.box-solid.) treated with Fe(COMPOUND I).sub.3.
[0035] FIG. 15 shows the Induction of KLF4 and CDKN1A (p21) by
COMPOUND I in AML cell lines. A) KLF4 mRNA induction after 24 hr
treatment with COMPOUND I at concentrations listed. B)
Concentration-dependent increase in CDKN1A mRNA expression in AML
lines. C) Western blot analysis of CDKN1A protein level in MV4-11
cells after 24 h exposure to vehicle (v) or increasing
concentrations of COMPOUND I. D) Time dependent increase in p21
mRNA. E) Western blot analysis of CDKN1A protein level in MV4-11
cells as a function of duration of exposure to 500 nM COMPOUND I
(A) as compared with vehicle (V). All mRNA measurements were made
by qRT-PCR and graphed relative to GAPDH loading control. Error
bars, .+-.SD from 3 replicate experiments.
[0036] FIG. 16 shows that COMPOUND I induces G.sub.0/G.sub.1 cell
cycle arrest in a dose- and time-dependent manner in AML cell
lines. A) Representative western blot of CDK4 and CCND3 protein
level in MV4-11, KG-1, and EOL-1 cells quantitated in lower panels
of FIGS. 16A-C. B) CDK4 and CCND3 protein levels after exposure to
IC.sub.50 concentration of COMPOUND I for the times noted. Protein
levels quantitated from 3 independent western blots, graphed as
fold change over vehicle. Error bars, .+-.SD. C) Representative
western blot of CDK4 and CCND3 protein levels as a function of
duration of COMPOUND I exposure (MV4-11 cells 500 nM, KG-1 cells
600 nM, and EOL-1 cells 300 nM). V--vehicle, A--COMPOUND I.
[0037] FIG. 17 shows that COMPOUND I treatment induces apoptosis in
a time- and concentration-dependent manner A) Histograms showing
distribution of early versus late apoptotic cells. B) Total
apoptotic cells in COMPOUND I versus vehicle treated MV4-11, KG-1,
and EOL-1 cells as a function of time. Error bars, .+-.SD from 2
replicate experiments.
[0038] FIG. 18 shows the pathways regulated by COMPOUND I in MV4-11
cells. A) Gene Ontology analysis (GO) of differentially expressed
genes detected by RNA-seq analysis of MV4-11 cells treated with
vehicle or 500 nM COMPOUND I for 6 h. GO terms and p-values were
computed using Broad Molecular Signatures database (MSigDB). B)
Normalized protein levels in vehicle and COMPOUND I (500 nM)
treated MV4-11 cells after 6 h. Protein levels detected by Reverse
Phase Protein Array. Heat-map generated in GraphPad Prism, average
of 3 replicate samples. C) GO analysis of differentially expressed
proteins utilizing MSigDB.
[0039] FIG. 19 shows the regulation of MYC expression by COMPOUND I
in AML cells. A) Total MYC protein levels in MV4-11, KG-1, and
EOL-1 cells treated with 500 nM COMPOUND I for the times noted.
Protein levels quantitated from 3 independent western blots,
normalized to GAPDH and graphed as fold change over vehicle. Error
bars, .+-.SD. B) Representative western blot quantitated in A). C)
Basal protein expression of MYC in AML cell lines versus PBMCs from
healthy donors. D) Position of MYC specific primer pairs used in
ChIP-qPCR analysis. E) ChIP-qPCR analysis of H3K27ac at MYC
promoter in MV4-11 cells treated with 500 nM COMPOUND I for 2, 6,
and 24 h graphed as fold change over vehicle treated after
normalization to input. F) MYC mRNA level assayed by RT-qPCR in
MV4-11 cells pretreated with COMPOUND I (500 nM) or vehicle for 3
h. Samples taken at time points listed after addition of 1 .mu.M
actinomycin D. Error bars, .+-.SD from 3 biological replicates
experiments.*P-value<0.05, ** <0.005, calculated by TTEST
using excel.
[0040] FIG. 20 shows the cellular pharmacology of COMPOUND I. A)
Time course of COMPOUND I uptake into KG-1 cells. B) Efflux of
COMPOUND I from cells loaded for either 1 or 6 h by exposure of
KG-1 cells to 1 .mu.M COMPOUND I. C) Structure of parental
monomeric COMPOUND I. D) Structure of Fe(COMPOUND I).sub.3. E)
COMPOUND I and Fe(COMPOUND I).sub.3 uptake into MV4-11 cells.
[0041] FIG. 21 shows the FRET assay analysis of G-quadruplex
structures. A) Schematic of quenching FRET assay. At low
temperatures, the G-quadruplex structure forms and the fluorescent
FAM signal is quenched by BHQ1; as the temperature is increased the
G4 structure unfolds and the FAM signal increases. Temperature at
which fluorescent signal is 50% of max (T.sub.1/2) was calculated
for each drug concentration then the .DELTA.T.sub.1/2 (drug
T.sub.1/2-Vehicle T.sub.1/2) was plotted against drug
concentration. B) Melting curves of ds-DNA control oligos. C)
Melting curves for G4 oligos after 6 hr incubation with COMPOUND I.
Error bars, .+-.SD from 3 biological replicates experiments.
[0042] FIG. 22 shows that Fe(COMPOUND I).sub.3 stabilizes Tm of
G-quadruplex oligos. A-D) Melting curves of 5' FAM-3' BHQ1 dual
labeled oligos containing G-quadruples sequences for A) Human
telomeres, B) MYC gene promoter, C) rRNA loci, and D) KIT gene
promoter. Error bars, .+-.SD from 3 biological replicates
experiments.
DETAILED DESCRIPTION
[0043] In view of the foregoing challenges relating to the
identification of drugs for which loss of BRCA1 or BRCA2 function
results in cellular hypersensitivity but that do not cause
myelosuppression in an individual, COMPOUND I has been identified.
It was unexpectedly discovered that COMPOUND I causes DNA damage,
and cells deficient in homologous recombination are as
hypersensitive to this drug as they are to olaparib, which is an
FDA-approved PARP inhibitor. COMPOUND I joins the limited
repertoire of drugs which can exploit defects in homologous
recombination while not causing myelosuppression.
[0044] Mechanistic studies on the mechanisms of action and
resistance to COMPOUND I were also undertaken, so as to identify
synthetic lethal interactions that can guide combination drug
studies. As described herein, COMPOUND I is converted
intracellularly into an Fe complex (Fe(COMPOUND I).sub.3) which is
an active form of the drug. COMPOUND I generated DNA damage at
early time points as documented by .gamma.H2AX accumulation and
foci formation. BRCA1- and BRCA2-deficient cells were found to be
hypersensitive to COMPOUND I to a degree comparable to that of
olaparib. Resistance to COMPOUND I in Raji cells is associated with
up-regulation of the efflux transporter ABCG2 and resistance is
partially reversed by ABCG2 inhibition. The ability of COMPOUND I
to exploit homologous recombination deficiency is of particular
interest because, unlike all the other drugs for which loss of this
repair function results in hypersensitivity, COMPOUND I does not
produce myelosuppression even at the maximum tolerated dose.
[0045] COMPOUND I is of interest because it is a member of a novel
class of compounds that exhibits potent cytotoxicity against a wide
range of both solid tumor and hematologic malignancies and does not
cause myelosuppression. The key findings reported herein are that
the COMPOUND I monomer can be converted intracellularly to an
active complex containing a ferrous Fe atom and three molecules of
COMPOUND I, whose intracellular concentration may exceed that of
the native drug. COMPOUND I and/or its complex with iron causes DNA
damage, in which the DNA repair requires the function of both BRCA1
and BRCA2 as evidenced by synthetic lethality with COMPOUND I. In
the case of Raji lymphoma cells, acquired resistance is associated
with reduced drug uptake and marked over-expression of the ABCG2
drug efflux pump whose inhibition partially reverses
resistance.
[0046] Compared with many other chemotherapeutic agents used to
treat lymphoma, the cellular accumulation of COMPOUND I is
relatively slow, but it appears to be rapidly converted to
Fe(COMPOUND I).sub.3 as this complex is present as soon as the
native form of the drug is detected in the cell. By 6 h the
cellular content of the Fe(COMPOUND I).sub.3 exceeded that of the
native form by .about.18-fold. The potency of the Fe(COMPOUND
I).sub.3 complex is only 2-fold less than that of native drug which
can be accounted for by the fact that, while COMPOUND I is neutral,
Fe(COMPOUND I).sub.3 is much larger and contains a 2.sup.+ charge
which would be expected to impair transmembrane influx. The fact
that no native drug was detectable in cells incubated with the
Fe(COMPOUND I).sub.3 complex strongly suggests that Fe(COMPOUND
I).sub.3 is an active intracellular form of the drug. Drugs
containing the 2,10 indole ring structure are known to chelate Fe
and Zn. In the case of COMPOUND I, while the Fe chelate was
abundant in cells, a Zn chelate was not detectable. Indeed, the Fe
chelate levels were high enough that cells exposed to COMPOUND I
became pink in color. The high level of Fe(COMPOUND I).sub.3 raises
the question of whether its formation depletes cells of Fe to the
point where cellular metabolism is impaired and this remains an
interesting point for further investigation. Without being bound by
any particular theory, chelation may be facilitated by the
intracellular environment, as no extracellular Fe(COMPOUND I).sub.3
was detected when COMPOUND I was incubated with complete tissue
culture medium.
[0047] The observation that deficiency in homologous recombination
produced by loss of BRCA1/2 function results in hypersensitivity to
certain types of DNA damaging drugs has been exploited to increase
the effectiveness of the platinum-containing drugs cisplatin and
carboplatin, and the PARP inhibitors olaparib and niraparib
particularly in the case of ovarian cancer. Ledermann et al.,
"Olaparib Maintenance Therapy in Platinum-Sensitive Relapsed
Ovarian Cancer," N. Engl. J. Med., 2012; 366 (15):1382-92; Mirza et
al., "Niraparib Maintenance Therapy in Platinum-Sensitive,
Recurrent Ovarian Cancer," N. Engl. J. Med., 2016; 375
(22):2154-64, both of which are incorporated by reference. Various
degrees of homologous recombination deficiency have been identified
at lower frequency in a variety of other tumors. Davies et al.,
"HRDetect is a Predictor of BRCA1 and BRCA2 Deficiency Based on
Mutational Signatures," Nat. Med. 2017; 23 (4):517-525, which is
hereby incorporated by reference. The ability of COMPOUND I to
exploit homologous recombination deficiency is of particular
interest because, unlike all the other drugs for which loss of this
repair function results in hypersensitivity, COMPOUND I does not
produce myelosuppression even at the maximum tolerated dose. Cercek
et al., "Phase 1 study of COMPOUND I HCl, an Inducer of KLF4, in
Patients with Advanced or Metastatic Solid Tumors," Invest. New
Drugs, 2015; 33 (5):1086-92, which is hereby incorporated by
reference. Thus, COMPOUND I joins the limited repertoire of drugs
which can take advantage of this important therapeutic window. The
observations reported herein identify .gamma.-H2AX as a potential
biomarker of clinical drug effect and point the way toward more
detailed studies of how COMPOUND I causes DNA damage. Ivashkevich
et al., "Use of the Gamma-H2AX Assay to Monitor DNA Damage and
Repair in Translational cancer Research," Cancer Lett, 2012; 327
(1-2):123-33, which is hereby incorporated by reference.
[0048] Development of acquired resistance to COMPOUND I in the Raji
lymphoma cells was associated with reduced accumulation of COMPOUND
I and the Fe(COMPOUND I).sub.3 complex. There was 16.5.+-.1.94 fold
more intracellular Fe(COMPOUND I).sub.3 in the Raji sensitive cells
than the resistant cells which corresponds perfectly to the
relative resistance of the Raji/COMPOUND IR over the sensitive
cells (16.7.+-.3.9 fold). RNA-seq analysis of the Raji/COMPOUND IR
cells pointed most directly to over-expression of ABCG2 as a
possible mechanism of resistance. Western blot analysis confirmed
up-regulation at the protein level, and that ABCG2 was functional
and directly involved in COMPOUND I resistance was established by
the ability of its inhibitor to partially reverse resistance to
COMPOUND I as well as topotecan. The fact that accumulation of
Fe(COMPOUND I).sub.3 was reduced in the resistant cells incubated
with the pre-formed complex suggests that the Fe(COMPOUND I).sub.3
as well as the native drug may be a substrate for the ABCG2
transporter. None of the known classes of drugs for which increased
ABCG2 confers resistance have obvious structural similarity to
COMPOUND I or Fe(COMPOUND I).sub.3. Thus, the discovery that ABCG2
can mediate resistance to COMPOUND I expands the range of known
substrates for this important transporter. Whether ABCG2 can be
used as a biomarker for sensitivity to COMPOUND I will need to be
explored in a large panel of cell lines. A search of the
Connectivity Map (https://portals.broadinstitute.org/cmap/) did not
disclose any significant similarity between the cytotoxicity
pattern of COMPOUND I and any of the other drugs thus far tested in
the large panel of cell lines further highlighting the uniqueness
of this compound.
[0049] Given that the specific inhibitor of ABCG2, Ko143, did not
completely reverse acquired COMPOUND I resistance, it seems likely
that other mechanisms also contribute to the phenotype. In this
regard, the cross-resistance to carboplatin is of particular
interest. Carboplatin is not a known ABCG2 substrate, but it too
causes DNA damage and up-regulation of transcription-coupled repair
has been widely reported to contribute to resistance to both
carboplatin and cisplatin, both of which produce the same types of
adducts in DNA. Enoiu et al., "Repair of Cisplatin-induced DNA
Interstrand Crosslinks by a Replication-independent Pathway
Involving Transcription-coupled Repair and Translesion Synthesis,"
Nucleic Acids Res. 2012; 40 (18):8953-64, which is hereby
incorporated by reference. It remains to be determined whether
up-regulation of DNA repair capacity contributes to both
carboplatin and COMPOUND I resistance.
[0050] Also described herein, it was discovered that COMPOUND I is
associated with CDKN1A upregulation and MYC downregulation,
followed by G.sub.0-G.sub.1 cell-cycle arrest and apoptosis in AML
cells. Moreover, inhibition of MYC, a well-recognized pivotal
oncogene in AML, correlated with the cytotoxicity of COMPOUND I.
Differential expression analysis suggested the involvement of DNA
damage, including induction of .gamma.-H2AX accumulation, and
cellular stress pathways after COMPOUND I treatment. Prior cellular
pharmacokinetic studies demonstrated that COMPOUND I is transformed
from a monomeric form to a ferrous complex [Fe(COMPOUND I).sub.3]
in cells, and that this complex is the principal intracellular form
of the drug. In this study, we demonstrate that the parental
COMPOUND I and the Fe(COMPOUND I).sub.3 complex bind to and
stabilize G-quadruplex (G4) motifs. The Fe(COMPOUND I).sub.3
complex stabilized G4 motifs found in the promoters of key
oncogenes (e.g., MYC, KIT), as well as in rRNA genes and telomeres.
This stabilization of secondary DNA structures was specific for G4
motifs, as the parental COMPOUND I and Fe(COMPOUND I).sub.3 did not
interact with dsDNA. Treatment of MV4-11 AML cells with preformed
Fe(COMPOUND I).sub.3 also inhibits MYC expression and induces
CDKN1A expression along with induction of apoptotic and DNA damage
pathways. Together, the results support the conclusion that the
effect of COMPOUND I on the expression of MYC and its downstream
target genes, on cell-cycle arrest, and on DNA damage and stress
responses can be linked to the action of COMPOUND I and the
Fe(COMPOUND I).sub.3 on G-quadruplex DNA motifs.
Definitions
[0051] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which the present application belongs.
Although any methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present application, representative methods and materials are
herein described.
[0052] Reference throughout this specification to "one embodiment"
or "an embodiment" means that a particular feature, structure or
characteristic described in connection with the embodiment is
included in at least one embodiment. Thus, the appearances of the
phrases "in one embodiment" or "in an embodiment" in various places
throughout this specification are not necessarily all referring to
the same embodiment. Furthermore, the particular features,
structures, or characteristics can be combined in any suitable
manner in one or more embodiments. Also, as used in this
specification and the appended claims, the singular forms "a,"
"an," and "the" include plural referents unless the content clearly
dictates otherwise. It should also be noted that the term "or" is
generally employed in its sense including "and/or" unless the
content clearly dictates otherwise.
[0053] Unless otherwise indicated, all numbers expressing
quantities of ingredients, reaction conditions, and so forth used
in the specification and claims are to be understood as being
modified in all instances by the term "about". Accordingly, unless
indicated to the contrary, the numerical parameters set forth in
the present specification and attached claims are approximations
that can vary depending upon the desired properties sought to be
obtained by the present application.
[0054] Throughout the present specification, numerical ranges are
provided for certain quantities. It is to be understood that these
ranges comprise all subranges therein. Thus, the range "from 50 to
80" includes all possible ranges therein (e.g., 51-79, 52-78,
53-77, 54-76, 55-75, 60-70, etc.). Furthermore, all values within a
given range can be an endpoint for the range encompassed thereby
(e.g., the range 50-80 includes the ranges with endpoints such as
55-80, 50-75, etc.).
[0055] COMPOUND I refers to
2-(5-fluoro-2-methyl-1H-indol-3-yl)-1H-imidazo[4,5-f][1,10]phenanthroline-
, pharmaceutically acceptable salts, esters, prodrugs, hydrates,
solvates and isomers thereof, for the structure below.
##STR00008##
[0056] Fe(COMPOUND I).sub.3 refers to the following structure:
##STR00009##
[0057] A "pharmaceutically acceptable salt" includes both acid and
base addition salts.
[0058] A pharmaceutically acceptable salt of COMPOUND I may be a
"pharmaceutically acceptable acid addition salt" derived from
inorganic or organic acid, and such salt may be pharmaceutically
acceptable nontoxic acid addition salt containing anion. For
example, the salt may include acid addition salts formed by
inorganic acids such as hydrochloric acid, sulfuric acid, nitric
acid, phosphoric acid, hydrobromic acid, hydroiodic acid, and the
like; organic carbonic acids such as tartaric acid, formic acid,
citric acid, acetic acid, trichloroacetic acid, trifluoroacetic
acid, gluconic acid, benzoic acid, lactic acid, fumaric acid,
maleic acid, and the like; and sulfonic acids such as
methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid,
naphthalensulfonic acid, and the like.
[0059] The pharmaceutically acceptable salt of COMPOUND I may be
prepared by conventional methods well-known in the art.
Specifically, the "pharmaceutically acceptable salt" in accordance
of the present invention may be prepared by, e.g., dissolving
COMPOUND I in a water-miscible organic solvent such as acetone,
methanol, ethanol or acetonitrile and the like; adding an excessive
amount of organic acid or an aqueous solution of inorganic acid
thereto; precipitating or crystallizing the mixture thus obtained.
Further, it may be prepared by further evaporating the solvent or
excessive acid therefrom; and then drying the mixture or filtering
the extract by using, e.g., a suction filter.
[0060] The term "chelate" as used herein means a molecular entity
made up of a central metal associated with at least one bidentate
ligand and optionally associated with one or more mono- or
multi-dentate ligands. For example, a "chelate" as used means a
molecular entity made up of a central metal associated with at
least one bidentate ligand of COMPOUND I. In the interaction
between the central metal and any of the ligands, the bonds between
the ligand and the central metal can include covalent bonds, ionic
bonds, and/or coordinate covalent bonds.
[0061] The term "complex" or "metal complex" as used herein means a
coordination complex of a metal and a ligand. For example, a
"complex" or "metal complex" as used herein means a coordination
complex of a metal and COMPOUND I.
[0062] The term "metal" as used herein means any alkaline, alkaline
earth, transition, rare earth, basic, and semi-metals which can
coordinate with a ligand. Representative metals include the
transition metals, lanthanide, and actinide metals. In some
embodiments, the metal has d-orbitals capable of interacting with a
ligand. For example, the metal may be iron, zinc, aluminum,
magnesium, platinum, silver, gold, chromium, nickel, titanium,
copper, scandium, zirconium, vanadium, molybdenum, manganese,
tungsten and cobalt. In one embodiment, the metal is iron.
[0063] The term "ester" as used herein refers to a chemical moiety
having chemical structure of --(R).sub.n--COOR', wherein R and R'
are each independently selected from the group consisting of alkyl,
cycloalkyl, aryl, heteroaryl (connected to oxygen atom by aromatic
ring) and heteroalicyclic (connected by aromatic ring), and n is 0
or 1, unless otherwise indicated.
[0064] The term "prodrug" as used herein refers to a precursor
compound that will undergo metabolic activation in vivo to produce
the parent drug. Prodrugs are often useful because they can be
easily administered as compared to parent drugs thereof in some
cases. For instance, some prodrugs are bioavailable via oral
administration unlike parent drugs thereof often show poor
bioavailability. Further, the prodrugs may show improved solubility
in the pharmaceutical composition as compared to parent drugs
thereof. For instance, COMPOUND I may be administered in the form
of an ester prodrug so as to increase drug delivery efficiency
since the solubility of a drug can adversely affect the
permeability across the cell membrane. Then, once the compound in
the form of the ester prodrug enters a target cell, it may be
metabolically hydrolyzed into a carboxylic acid and an active
entity.
[0065] Hydrates or solvates of COMPOUND I are included within the
scope of the present invention. As used herein, "solvate" means a
complex formed by solvation (the combination of solvent molecules
with molecules or ions of the active agent of the present
invention), or an aggregate that consists of a solute ion or
molecule (the active agent of the present invention) with one or
more solvent molecules. The solvent can be water, in which case the
solvate can be a hydrate. Examples of hydrate include, but are not
limited to, hemihydrate, monohydrate, dihydrate, trihydrate,
hexahydrate, etc. It should be understood by one of ordinary skill
in the art that the pharmaceutically acceptable salt of the present
compound may also exist in a solvate form. The solvate is typically
formed via hydration which is either part of the preparation of the
present compound or through natural absorption of moisture by the
anhydrous compound of the present invention. Solvates including
hydrates may be consisting in stoichiometric ratios, for example,
with two, three, four salt molecules per solvate or per hydrate
molecule. Another possibility, for example, that two salt molecules
are stoichiometric related to three, five, seven solvent or hydrate
molecules. Solvents used for crystallization, such as alcohols,
especially methanol and ethanol; aldehydes; ketones, especially
acetone; esters, e.g. ethyl acetate; may be embedded in the crystal
grating particularly pharmaceutically acceptable solvents.
[0066] The compounds of the disclosure or their pharmaceutically
acceptable salts can contain one or more axes of chirality such
that atropisomerization is possible. Atropisomers are stereoisomers
arising because of hindered rotation about a single bond, where
energy differences due to steric strain or other contributors
create a barrier to rotation that is high enough to allow for
isolation of individual conformers. The present disclosure is meant
to include all such possible isomers, as well as their racemic and
optically pure forms whether or not they are specifically depicted
herein. Optically active isomers can be prepared using chiral
synthons or chiral reagents, or resolved using conventional
techniques, for example, chromatography and fractional
crystallization. Conventional techniques for the
preparation/isolation of individual atropisomers include chiral
synthesis from a suitable optically pure precursor or resolution of
the racemate (or the racemate of a salt or derivative) using, for
example, chiral high pressure liquid chromatography (HPLC).
[0067] A "stereoisomer" refers to a compound made up of the same
atoms bonded by the same bonds but having different
three-dimensional structures, which are not interchangeable. The
present invention contemplates various stereoisomers and mixtures
thereof as it pertains to atropisomerism.
[0068] The terms "treat", "treating" or "treatment" in reference to
a particular disease or disorder includes prevention of the disease
or disorder, and/or lessening, improving, ameliorating or
abrogating the symptoms and/or pathology of the disease or
disorder. Generally, the terms as used herein refer to
ameliorating, alleviating, lessening, and removing symptoms of a
disease or condition. COMPOUND I herein may be in a therapeutically
effective amount in a formulation or medicament, which is an amount
that can lead to a biological effect, such as DNA damage, apoptosis
of certain cells (e.g., cancer cells), reduction of proliferation
of certain cells, or lead to ameliorating, alleviating, lessening,
or removing symptoms of a disease or condition, for example. The
terms also can refer to reducing or stopping a cell proliferation
rate (e.g., slowing or halting tumor growth) or reducing the number
of proliferating cancer cells (e.g., removing part or all of a
tumor).
[0069] When treatment as described above refers to prevention of a
disease, disorder, or condition, said treatment is termed
prophylactic. Administration of said prophylactic agent can occur
prior to the manifestation of symptoms characteristic of a
proliferative disorder, such that a disease or disorder is
prevented or, alternatively, delayed in its progression.
[0070] As used herein, the terms "inhibiting" or "reducing" cell
proliferation is meant to slow down, to decrease, or, for example,
to stop the amount of cell proliferation, as measured using methods
known to those of ordinary skill in the art, by, for example, 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, when compared
to proliferating cells that are not subjected to the methods,
compositions , and combinations of the present application.
[0071] As used herein, "cell cycle arrest" refers to the halting of
a series of events that take place in the cell leading to its
division and replication, which may be caused by a number of
factors, including, but not limited to, DNA damage, X-radiation,
ionizing radiation, and chemotherapeutic agents. In certain
embodiments, "DNA damage" and "cell cycle arrest" are used
interchangeably.
[0072] As used herein, the term "apoptosis" refers to an intrinsic
cell self-destruction or suicide program. In response to a
triggering stimulus, cells undergo a cascade of events including
cell shrinkage, blebbing of cell membranes and chromatic
condensation and fragmentation. These events culminate in cell
conversion to clusters of membrane-bound particles (apoptotic
bodies), which are thereafter engulfed by macrophages.
[0073] As used herein, "myelosuppression" refers to the suppression
of one or more components of hematopoiesis, which manifests in
aberrant levels of one or more of the cell types that are the
products of this process. For a review of hematopoiesis, and
characteristics of hematopoietic cells, see Clinical Immunology:
Principles and Practice, Vol. 1, Ch. 2, pp. 15-24 (Lewis and
Harriman, eds. Mosby--Year Book, Inc. 1996), which pages are hereby
incorporated by reference. On a general level, it refers to
decreases in white blood cell and/or platelet counts. It also
refers, on a more specific level, to suppression, relative to
normal levels, of one or more of the following cells that result
from hematopoiesis: B-cells, T-cells, natural killer cells,
dendritic cells, macrophages, neutrophils, eosinophils, basophils,
mast cells and platelets. Other terms may be used interchangeably
with myelosuppression and will be readily apparent to a skilled
artisan. Non-limiting examples of such terms include "bone marrow
suppression," "myelotoxicity," and myeloablation." On the other
hand, therefore, "myelorecovery" is the opposite of
myelosuppression. Therefore, in one embodiment, the term "bone
marrow activity" refers to the levels of the following cells that
result from hematopoiesis: B-cells, T-cells, natural killer cells,
dendritic cells, macrophages, neutrophils, eosinophils, basophils,
mast cells platelets, erythrocytes, platelets, myeloid and lymphoid
white blood cells and others that are apparent to a skilled
artisan.
[0074] The term "subject" as used herein, refers to an animal, such
as a mammal or non-mammal. For example, the subject may be a
mammal, such as a human, who is in the need of treatment or
prevention of cancer. The term subject may be interchangeably used
with the term patient in the context of the present invention.
[0075] "Mammal" includes humans and both domestic animals such as
laboratory animals and household pets (e.g., cats, dogs, swine,
cattle, sheep, goats, horses, rabbits), and non-domestic animals
such as wildlife and the like. The term "patient" or "subject" as
used herein, includes humans and animals.
[0076] "Non-mammal" includes a lion-mammalian invertebrate and
non-mammalian vertebrae, such as a bird (e.g., a chicken or duck)
or a fish.
[0077] A "pharmaceutical composition" refers to a formulation of a
compound of the disclosure and a medium generally accepted in the
art for the delivery of the biologically active compound to
mammals, e.g., humans. Such a medium includes all pharmaceutically
acceptable carriers, diluents or excipients therefor.
[0078] "An "effective amount" refers to a therapeutically effective
amount or a prophylactically effective amount. A "therapeutically
effective amount" refers to an amount effective, at dosages and for
periods of time necessary, to achieve the desired therapeutic
result, such as cancer cell death, reduced tumor size, increased
life span or increased life expectancy. A therapeutically effective
amount of a compound can vary according to factors such as the
disease state, age, sex, and weight of the subject, and the ability
of the compound to elicit a desired response in the subject. Dosage
regimens can be adjusted to provide the optimum therapeutic
response. A therapeutically effective amount is also one in which
any toxic or detrimental effects of the compound are outweighed by
the therapeutically beneficial effects. A "prophylactically
effective amount" refers to an amount effective, at dosages and for
periods of time necessary, to achieve the desired prophylactic
result, such as smaller tumors or slower cell proliferation.
Typically, a prophylactic dose is used in subjects prior to or at
an earlier stage of disease, so that a prophylactically effective
amount can be less than a therapeutically effective amount.
Methods
[0079] The present invention provides methods of preventing,
reducing, or treating cancer in a subject.
[0080] In one embodiment of the present disclosure, a method is
provided for preventing, reducing, or treating cancer in a subject,
comprising administering a therapeutically effective amount of
##STR00010##
(COMPOUND I) or a pharmaceutically acceptable salt, free base,
hydrate, complex, or chelate (including metal chelates, such as
iron, zinc and others) thereof to the subject, wherein the subject
has a mutation in a DNA repair gene. In an embodiment, the subject
is a human. In another embodiment, the subject already has
cancer.
[0081] In another embodiment, the present disclosure relates to a
method of preventing, reducing or treating cancer in a subject,
comprising administering a therapeutically effective amount of one
or more molecules of COMPOUND I in complex with one or more metal
atoms, wherein the subject has a mutation in a DNA repair gene. In
one embodiment, the one or more metal atoms are selected from the
group consisting of iron, zinc, aluminum, magnesium, platinum,
silver, gold, chromium, nickel, titanium, copper, scandium,
zirconium, vanadium, molybdenum, manganese, tungsten and cobalt. In
one embodiment, the one or more metal atoms are iron. In certain
embodiments, the complex has the following structure:
##STR00011##
[0082] In an embodiment, the DNA repair gene is a homologous
recombinant gene. In certain embodiments, the DNA repair gene is a
gene in the homologous recombination (HR) dependent
deoxyribonucleic acid (DNA) double strand break (DSB) repair
pathway. A skilled artisan will appreciate that the HR dependent
DNA DSB repair pathway repairs double-strand breaks (DSBs) in DNA
via homologous mechanisms to reform a continuous DNA helix. K. K.
Khanna and S. P. Jackson, Nat. Genet. 27(3): 247-254 (2001), which
is hereby incorporated by reference in its entirety. The components
of the HR dependent DNA DSB repair pathway include, but are not
limited to, ATM, ATR, CHK1, CHK2, RPA, RAD51, RAD51L1, RAD51C,
RAD51L3, DMC1, XRCC2, XRCC3, RAD52, RAD54L, RAD54B, BRCA1, BRCA2,
RAD50, MRE11A and NBS1. Other proteins involved in the HR dependent
DNA DSB repair pathway include regulatory factors such as EMSY.
Hughes-Davies et al, Cell, Vol 115, pp 523-535, which is hereby
incorporated by reference in its entirety. Thus, in certain
embodiments, the DNA repair gene is one or more genes selected from
the group consisting of BRCA-1, BRCA-2, ATM, ATR, CHK1, CHK2,
Rad51, RPA, and XRCC3. In certain embodiments, the DNA repair gene
is BRCA-1 and/or BRCA-2.
[0083] In an embodiment of the present disclosure, the subject is
heterozygous for a mutation in a DNA repair gene. In certain
embodiments, the subject is heterozygous for a mutation in a gene
in the homologous recombination (HR) dependent deoxyribonucleic
acid (DNA) double strand break (DSB) repair pathway. Thus, in
certain embodiments, the gene in the homologous recombination (HR)
dependent deoxyribonucleic acid (DNA) double strand break (DSB)
repair pathway is one or more genes selected from the group
consisting of BRCA-1, BRCA-2, ATM, ATR, CHK1, CHK2, Rad51, RPA and
XRCC3. In certain embodiments, the DNA repair gene is BRCA-1 and/or
BRCA-2.
[0084] In an embodiment of the present disclosure, the subject is
homozygous for a mutation in a DNA repair gene. In certain
embodiments, the subject is homozygous for a mutation in a gene in
the homologous recombination (HR) dependent deoxyribonucleic acid
(DNA) double strand break (DSB) repair pathway. Thus, in certain
embodiments, the gene in the homologous recombination (HR)
dependent deoxyribonucleic acid (DNA) double strand break (DSB)
repair pathway is one or more genes selected from the group
consisting of BRCA-1, BRCA-2, ATM, ATR, CHK1, CHK2, Rad51, RPA and
XRCC3. In certain embodiments, the DNA repair gene is BRCA-1 and/or
BRCA-2.
[0085] In an embodiment, the subject is administered a
therapeutically effective amount of COMPOUND I, or a
pharmaceutically acceptable salt, free base, hydrate, complex, or
chelate (including metal chelates, such as iron, zinc and others)
thereof for the treatment or prevention of cancer. A skilled
artisan will appreciate that within the context of the present
disclosure, a variety of cancers may be treated or prevented. Thus,
in an embodiment, the cancer is selected from the group consisting
of heme cancer, colorectal cancer, ovarian cancer, breast cancer,
cervical cancer, lung cancer, liver cancer, pancreatic cancer,
cancer of the lymph nodes, leukemia, renal cancer, colon cancer,
prostate cancer, brain cancer, cancer of the head and neck, bone
cancer, carcinoma of the larynx and oral cavity, Ewing's sarcoma,
skin cancer, kidney cancer, and cancer of the heart. In certain
embodiments, the cancer is selected from the group consisting of
breast cancer, lung cancer, cancer of the lymph nodes, colon
cancer, leukemia, renal cancer, and prostate cancer. In one
embodiment, the cancer is breast cancer. In some embodiments, the
cancer is a hematological malignancy. Examples of hematological
malignancies include, but are not limited to, leukemias, lymphomas,
Hodgkin's disease, and myeloma. Also, acute lymphocytic leukemia
(ALL), acute myeloid leukemia (AML), acute promyelocytic leukemia
(APL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia
(CML), chronic neutrophilic leukemia (CNL), acute undifferentiated
leukemia (AUL), anaplastic large-cell lymphoma (ALCL),
prolymphocytic leukemia (PML), juvenile myelomonocytic leukemia
(JMML), adult T-cell ALL, AML, with trilineage myelodysplasia
(AMLITMDS), mixed lineage leukemia (MLL), eosinophilic leukemia,
mantle cell lymphoma, myelodysplastic syndromes (MDSs) (e.g.
high-risk MDS), myeloproliferative disorders (MPD), and multiple
myeloma (MM). In some embodiments, the cancer is acute myeloid
leukemia. In some embodiments, the cancer is chronic myeloid
leukemia. In some embodiments, the cancer is a lymphoma. In some
embodiments, the cancer is high-risk myelodysplastic syndrome.
[0086] In an embodiment, the subject is administered a
therapeutically effective amount of COMPOUND I, or a
pharmaceutically acceptable salt, free base, hydrate, complex, or
chelate (including metal chelates, such as iron, zinc and others)
thereof for the treatment or prevention of a BRCA-associated
cancer. A skilled artisan will appreciate that a variety of cancers
are associated with BRCA. In an embodiment, the BRCA-associated
cancer has one or more mutations of the BRCA-1 and/or BRCA-2
genes.
[0087] The cancer cells may have a phenotype which is
characteristic of a deficiency in a component of HR dependent DNA
DSB repair pathway i.e. activity of a component of the pathway is
reduced or abolished in the cancer cells. Cancer cells with such a
phenotype may be deficient in a component of the pathway, for
example a component listed above i.e. expression and/or activity of
the component may be reduced or abolished in the cancer cells, for
example by means of mutation, polymorphism or epigenetic
modification, such as hypermethylation, in the encoding nucleic
acid or in a gene encoding a regulatory factor.
[0088] In some preferred embodiments, the cancer cells may have a
BRCA1 and/or a BRCA2 deficient phenotype i.e. BRCA1 and/or BRCA2
activity is reduced or abolished in the cancer cells. Cancer cells
with this phenotype may be deficient in BRCA1 and/or BRCA2 i.e.
expression and/or activity of BRCA1 and/or BRCA2 may be reduced or
abolished in the cancer cells, for example by means of mutation,
polymorphism or epigenetic modification, such as hypermethylation,
in the encoding nucleic acid or in a gene encoding a regulatory
factor, for example the EMSY gene which encodes a BRCA2 regulatory
factor (Hughes-Davies et al, Cell, Vol 115, pp 523-535, which is
hereby incorporated by reference).
[0089] BRCA1 and BRCA2 are known tumor suppressors whose wild-type
alleles are frequently lost in tumors of heterozygous carriers
(Jasin M. Oncogene. 2002 Dec. 16; 21(58):8981-93; Tutt et al Trends
Mol Med. (2002)8(12):571-6). The association of BRCA1 and/or BRCA2
mutations with breast cancer is well-characterized in the art
(Radice P J Exp Clin Cancer Res. 2002 September; 21 (3 Suppl):9-12,
which is hereby incorporated by reference). Amplification of the
EMSY gene, which encodes a BRCA2 binding factor, is also known to
be associated with breast and ovarian cancer.
[0090] Carriers of mutations in BRCA1 and/or BRCA2 are also at
elevated risk of cancer of the ovary, prostate and pancreas.
[0091] In other preferred embodiments, the cancer cells may have an
ATM, ATR, CHK1, CHK2, Rad51, DSS1, RPA and/or XRCC3 deficient
phenotype i.e. the activity of one or more of these components is
reduced or abolished in the cancer cells. Cancer cells may, for
example, be deficient in ATM, ATR, CHK1, CHK2, Rad51, DSS1, RPA
and/or XRCC3 i.e. expression and/or activity of ATM, ATR, CHK1,
CHK2, Rad51, DSS1, RPA and/or XRCC3 may be reduced or abolished in
the cancer cells, for example by means of mutation, polymorphism or
epigenetic modification, such as hypermethylation, in the encoding
nucleic acid or in a gene encoding a regulatory factor.
[0092] In an embodiment, the subject having a mutated DNA-repair
gene that is administered a therapeutically effective amount of
COMPOUND I, or a pharmaceutically acceptable salt, free base,
hydrate, complex, or chelate (including metal chelates, such as
iron, zinc and others) thereof is an animal. In certain
embodiments, the subject is a mammal. Thus, the subject within the
context of the present disclosure may be human, domestic animals
(e.g., laboratory animals), household pets (e.g., cats, dogs,
swine, cattle, sheep, goats, horses, rabbits), and non-domestic
animals such as wildlife and the like. In one embodiment, the
subject is a human.
Myelosuppression
[0093] In an embodiment, the method of the present disclosure is
directed to administering a therapeutically effective amount of
COMPOUND I, or a pharmaceutically acceptable salt, free base,
hydrate, complex, or chelate thereof to a subject, wherein the
incidence of myelosuppression in said subject is prevented or
lowered relative to a subject who was not administered a
therapeutically effective amount of COMPOUND I. In certain
embodiments, the subject who was not administered a therapeutically
effective amount of COMPOUND I has been administered a
chemotherapeutic agent that is not COMPOUND I for the treatment or
prevention of cancer. Thus, in one embodiment, the method of the
present disclosure is directed to administering a therapeutically
effective amount of COMPOUND I, or a pharmaceutically acceptable
salt, free base, hydrate, complex, or chelate (including metal
chelates, such as iron, zinc and others) thereof to a subject,
wherein the incidence of myelosuppression in said subject is
prevented or lowered relative to a subject who has been
administered a chemotherapeutic agent that is not COMPOUND I. As
used herein, myelosuppression generally refers to the suppression
of one or more components of hematopoiesis (e.g., bone marrow
activity), which manifests in aberrant levels of one or more of the
cell types that are the products of this process. The suppression
of one or more components of hematopoiesis (e.g., bone marrow
activity) may refer to, for example, the suppression of white blood
cell counts and/or platelet counts. Accordingly, in an embodiment,
a method of the present disclosure is provided for preventing,
reducing, or treating cancer in a subject, comprising administering
a therapeutically effective amount of COMPOUND I or a
pharmaceutically acceptable salt, free base, hydrate, complex, or
chelate (including metal chelates, such as iron, zinc and others)
thereof to the subject, wherein the subject has a mutation in a DNA
repair gene and wherein the subject experiences less than a 90%
decrease in bone marrow activity relative to a subject who was not
administered a therapeutically effective amount of COMPOUND I. For
instance, the subject experiences less than a 90%, 85%, 80%, 75%,
70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 24%, 23%, 22%,
21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%,
7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25% decrease in bone
marrow activity relative to a subject who was not administered a
therapeutically effective amount of COMPOUND I. In an embodiment,
the subject administered a therapeutically effective amount of
COMPOUND I experiences less than a 10% decrease in bone marrow
activity relative to a subject who was not administered a
therapeutically effective amount of COMPOUND I. In an embodiment,
the subject administered a therapeutically effective amount of
COMPOUND I experiences no decrease in bone marrow activity relative
to a subject who was not administered a therapeutically effective
amount of COMPOUND I.
[0094] In an embodiment, a method is provided for treating cancer
in a subject, comprising administering a therapeutically effective
amount of COMPOUND I, or a pharmaceutically acceptable salt, free
base, hydrate, complex, or chelate (including metal chelates, such
as iron, zinc and others) thereof to the subject, wherein the
subject has a mutation in a DNA repair gene. In certain
embodiments, various pathological conditions associated with
cancer, and which are readily apparent to a skilled artisan, may be
treated in a subject having cancer by administering a
therapeutically effective amount of COMPOUND I or a
pharmaceutically acceptable salt, free base, hydrate, complex, or
chelate (including metal chelates, such as iron, zinc and others)
thereof. Accordingly, in one embodiment, the subject experiences a
reduction or decrease in size of a tumor associated with a cancer.
The reduction or decrease in tumor size may be anywhere from about
a 1% reduction or decrease in tumor size to about a 100% reduction
or decrease in tumor size, including all integers and ranges
therebetween. For instance, the reduction or decrease in tumor size
may be about 1%, about 5%, about 10%, about 15%, about 20%, about
25%, about 30%, about 35%, about 40%, about 45%, about 50%, about
55%, about 60%, about 65%, about 70%, about 75%, about 80%, about
85%, about 90%, about 95%, or about 100%. In one embodiment, the
subject experiences complete elimination of the tumor associated
with cancer (i.e., 100% reduction or decrease in tumor size). In
another embodiment, the subject experiences inhibition, decrease,
or reduction of neo-vascularization or angiogenesis in a tumor
associated with a cancer. The decrease or reduction of
neo-vascularization or angiogenesis in a tumor associated with a
cancer may be anywhere from about a 1% reduction or decrease in
neo-vascularization or angiogenesis to about a 100% reduction or
decrease in neo-vascularization or angiogenesis, including all
integers and ranges therebetween. For instance, the reduction or
decrease in neo-vascularization or angiogenesis may be about 1%,
about 5%, about 10%, about 15%, about 20%, about 25%, about 30%,
about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,
about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,
about 95%, or about 100%. In one embodiment, the subject
experiences complete reduction or decrease in neo-vascularization
or angiogenesis associated with cancer (i.e., 100% reduction or
decrease in neo-vascularization or angiogenesis).
[0095] In one embodiment, the present disclosure is directed to a
method for killing cancer cells, comprising contacting said cells
with a therapeutically effective amount of COMPOUND I, or a
pharmaceutically acceptable salt, free base, hydrate, complex, or
chelate (including metal chelates, such as iron, zinc and others)
thereof. In certain embodiments, the cancer cells have a deficiency
in one or more genes selected from the group consisting of BRCA-1,
BRCA-2, ATM, ATR, CHK1, CHK2, Rad51, RPA and XRCC3.
[0096] In one embodiment, the present disclosure relates to a
method for inducing cell cycle arrest in cancer cells, comprising
contacting said cells with a therapeutically effective amount
of
##STR00012##
or a pharmaceutically acceptable salt, free base, hydrate, complex,
or chelate (including metal chelates, such as iron, zinc and
others) thereof. In certain embodiments, the cancer cells have a
deficiency in one or more genes selected from the group consisting
of BRCA-1, BRCA-2, ATM, ATR, CHK1, CHK2, Rad51, RPA and XRCC3.
[0097] In one embodiment, a method for stabilizing G-quadruplexes
(G4s) in a subject is provided where the method comprises
administering to the subject a therapeutically effective amount of
COMPOUND I, or a pharmaceutically acceptable salt, free base,
hydrate, complex, or chelate (including metal chelates, such as
iron, zinc and others) thereof. In another embodiment, a method for
stabilizing G-quadruplexes (G4s) in a subject is provided where the
method comprises administering to the subject a therapeutically
effective amount of a pharmaceutical combination comprising
COMPOUND I, or a pharmaceutically acceptable salt, free base,
hydrate, complex, or chelate (including metal chelates, such as
iron, zinc and others) thereof, and at least one additional
therapeutically active agent, as described herein. In some
embodiments, a method for stabilizing G-quadruplexes (G4s) in a
subject is provided where the method comprises administering to the
subject a therapeutically effective amount of a pharmaceutical
combination comprising COMPOUND I, or a pharmaceutically acceptable
salt, free base, hydrate, complex, or chelate (including metal
chelates, such as iron, zinc and others) thereof, and administering
radiotherapy or at least one additional therapeutically active
agent before, during, or after the subject has been administered
the aforementioned compound.
[0098] In one embodiment, COMPOUND I, or a pharmaceutically
acceptable salt, free base, hydrate, complex, or chelate (including
metal chelates, such as iron, zinc and others) thereof, is
administered at a dose from about 1 mg/day to about 3 g/day. In
certain embodiments, COMPOUND I, or a pharmaceutically acceptable
salt, free base, hydrate, complex, or chelate (including metal
chelates, such as iron, zinc and others) thereof, is administered
at a dose from about 1 mg/day to about 200 mg/day. In certain
embodiments, COMPOUND I, or a pharmaceutically acceptable salt,
free base, hydrate, complex, or chelate (including metal chelates,
such as iron, zinc and others) thereof, is administered at a dose
from about 50 mg/day to about 200 mg/day.
Combination Therapy
[0099] In one embodiment, the present invention provides a
combination therapy comprising COMPOUND I with at least one
additional therapeutically active agent.
[0100] In one embodiment, the present invention provides a method
of treating a condition associated with cell proliferation in a
patient in need thereof. In one embodiment, the present invention
provides a method of treating cancer or tumors. The method
comprises co-administering to a patient in need thereof a
therapeutically effective amount of COMPOUND I, or a
pharmaceutically acceptable salt, free base, hydrate, ester,
solvate and/or prodrug thereof and at least one additional
therapeutically active agent. In one embodiments, at least one
additional therapeutically active agent is Olaparib.
[0101] The term "co-administration" or "coadministration" refers to
administration of (a) COMPOUND I, or a pharmaceutically acceptable
salt, free base, hydrate, complex, or chelate (including metal
chelates, such as iron, zinc and others) thereof and (b) at least
one additional therapeutically active agent, together in a
coordinated fashion. For example, the co-administration can be
simultaneous administration, sequential administration, overlapping
administration, interval administration, continuous administration,
or a combination thereof. In one embodiment, COMPOUND I, or a
pharmaceutically acceptable salt, free base, hydrate, complex, or
chelate (including metal chelates, such as iron, zinc and others)
thereof and at least one additional therapeutically active agent
are formulated into a single dosage form. In another embodiment,
COMPOUND I, or a pharmaceutically acceptable salt, free base,
hydrate, complex, or chelate (including metal chelates, such as
iron, zinc and others) thereof and at least one additional
therapeutically active agent are provided in a separate dosage
forms.
[0102] Pharmaceutical Formulations
[0103] In another embodiment, the present invention provides a
pharmaceutical composition and/or combination comprising a
therapeutically effective amount of COMPOUND I or a
pharmaceutically acceptable salt, free base, hydrate, complex, or
chelate (including metal chelates, such as iron, zinc and others)
thereof, as disclosed herein, as the active ingredient, combined
with a pharmaceutically acceptable excipient or carrier. The
excipients are added to the formulation for a variety of
purposes.
[0104] In some embodiments, COMPOUND I or a pharmaceutically
acceptable salt, free base, hydrate, complex, or chelate (including
metal chelates, such as iron, zinc and others) thereof and at least
one therapeutically active agent may be formulated into a single
pharmaceutical composition and/or combination. In some embodiments,
COMPOUND I or a pharmaceutically acceptable salt, free base,
hydrate, complex, or chelate (including metal chelates, such as
iron, zinc and others) thereof and at least one therapeutically
active agent are formulated into a separate pharmaceutical
composition and/or combination comprising a pharmaceutically
acceptable excipient or a carrier.
[0105] In a specific embodiment, COMPOUND I or a pharmaceutically
acceptable salt, free base, hydrate, complex, or chelate (including
metal chelates, such as iron, zinc and others) thereof and at least
one therapeutically active agent may be formulated into a single
pharmaceutical composition and/or combination composition. In
another embodiment, the composition may comprise COMPOUND I or a
pharmaceutically acceptable salt, free base, hydrate, complex, or
chelate (including metal chelates, such as iron, zinc and others)
thereof, as disclosed herein, in an amount of about 1 mg to about 1
g. In another embodiment, the amount is about 5 mg to about 500 mg.
In another embodiment, the amount is about 20 mg to about 400 mg.
In another embodiment, the amount is about 50 mg to about 300 mg.
In another embodiment, the amount is about 100 mg to about 200 mg.
In another embodiment, the compound is a salt, ester, solvate or
prodrug of COMPOUND I.
[0106] In another embodiment, the pharmaceutical composition may
comprise a concentration of COMPOUND I or a pharmaceutically
acceptable salt, free base, hydrate, complex, or chelate (including
metal chelates, such as iron, zinc and others) thereof at about 0.1
mg/ml to about 10 mg/ml. In another embodiment, the concentration
is about 0.5 mg/ml to about 5 mg/ml. In another embodiment, the
concentration is about 0.75 mg/ml to about 4.5 mg/ml. In another
embodiment, the concentration is about at 3 mg/ml to about 5
mg/ml.
[0107] In another embodiment, the compound is a salt, ester,
solvate or prodrug of COMPOUND I. In another embodiment, the
composition may comprise COMPOUND I, or a pharmaceutically
acceptable salt, free base, hydrate, ester, solvate and/or prodrug
thereof, and a PARP inhibitor. In another embodiment, the PARP
inhibitor is Olaparib.
[0108] In another embodiment, the composition may comprise COMPOUND
I, or a pharmaceutically acceptable salt, free base, hydrate,
ester, solvate and/or prodrug thereof and Olaparib, wherein the
amount of Olaparib in the composition is about 10 mg to about 800
mg. In another embodiment, the amount of Olaparib is about 20 mg to
about 600 mg. In another embodiment, the amount of Olaparib is
about 100 mg to about 500 mg. In another embodiment, the amount of
Olaparib is about 300 mg to about 400 mg.
[0109] Pharmaceutical acceptable excipients may be added to the
composition/formulation. For example, diluents may be added to the
formulations of the present invention. Diluents increase the bulk
of a solid pharmaceutical composition and/or combination, and may
make a pharmaceutical dosage form containing the composition and/or
combination easier for the patient and care giver to handle.
Diluents for solid compositions and/or combinations include, for
example, microcrystalline cellulose (e.g., AVICEL), microfine
cellulose, lactose, starch, pregelatinized starch, calcium
carbonate, calcium sulfate, sugar, dextrates, dextrin, dextrose,
dibasic calcium phosphate dihydrate, tribasic calcium phosphate,
kaolin, magnesium carbonate, magnesium oxide, maltodextrin,
mannitol, polymethacrylates (e.g., EUDRAGIT(r)), potassium
chloride, powdered cellulose, sodium chloride, sorbitol, and
talc.
[0110] Solid pharmaceutical compositions and/or combinations that
are compacted into a dosage form, such as a tablet, may include
excipients whose functions include helping to bind the active
ingredient and other excipients together after compression. Binders
for solid pharmaceutical compositions and/or combinations include
acacia, alginic acid, carbomer (e.g., carbopol),
carboxymethylcellulose sodium, dextrin, ethyl cellulose, gelatin,
guar gum, gum tragacanth, hydrogenated vegetable oil, hydroxyethyl
cellulose, hydroxypropyl cellulose (e.g., KLUCEL), hydroxypropyl
methyl cellulose (e.g., METHOCEL), liquid glucose, magnesium
aluminum silicate, maltodextrin, methylcellulose,
polymethacrylates, povidone (e.g., KOLLIDON, PLASDONE),
pregelatinized starch, sodium alginate, and starch.
[0111] The dissolution rate of a compacted solid pharmaceutical
composition and/or combination in the patient's stomach may be
increased by the addition of a disintegrant to the composition
and/or combination. Disintegrants include alginic acid,
carboxymethylcellulose calcium, carboxymethylcellulose sodium
(e.g., AC-DI-SOL and PRIMELLOSE), colloidal silicon dioxide,
croscarmellose sodium, crospovidone (e.g., KOLLIDON and
POLYPLASDONE), guar gum, magnesium aluminum silicate, methyl
cellulose, microcrystalline cellulose, polacrilin potassium,
powdered cellulose, pregelatinized starch, sodium alginate, sodium
starch glycolate (e.g., EXPLOTAB), potato starch, and starch.
[0112] Glidants can be added to improve the flowability of a
non-compacted solid composition and/or combination and to improve
the accuracy of dosing. Excipients that may function as glidants
include colloidal silicon dioxide, magnesium trisilicate, powdered
cellulose, starch, talc, and tribasic calcium phosphate.
[0113] When a dosage form such as a tablet is made by the
compaction of a powdered composition and/or combination, the
composition and/or combination is subjected to pressure from a
punch and dye. Some excipients and active ingredients have a
tendency to adhere to the surfaces of the punch and dye, which can
cause the product to have pitting and other surface irregularities.
A lubricant can be added to the composition and/or combination to
reduce adhesion and ease the release of the product from the dye.
Lubricants include magnesium stearate, calcium stearate, glyceryl
monostearate, glyceryl palmitostearate, hydrogenated castor oil,
hydrogenated vegetable oil, mineral oil, polyethylene glycol,
sodium benzoate, sodium lauryl sulfate, sodium stearyl fumarate,
stearic acid, talc, and zinc stearate.
[0114] Flavoring agents and flavor enhancers make the dosage form
more palatable to the patient. Common flavoring agents and flavor
enhancers for pharmaceutical products that may be included in the
composition and/or combination of the present invention include
maltol, vanillin, ethyl vanillin, menthol, citric acid, fumaric
acid, ethyl maltol, and tartaric acid.
[0115] Solid and liquid compositions and/or combinations may also
be dyed using any pharmaceutically acceptable colorant to improve
their appearance and/or facilitate patient identification of the
product and unit dosage level.
[0116] In liquid pharmaceutical compositions and/or combinations
may be prepared using COMPOUND I, or a pharmaceutically acceptable
salt, free base, hydrate, ester, solvate and/or prodrug thereof, of
the present invention and any other solid excipients where the
components are dissolved or suspended in a liquid carrier such as
water, vegetable oil, alcohol, polyethylene glycol, propylene
glycol, glycerin, or macrogol 15 hydroxystearate (Solutol).
[0117] Liquid pharmaceutical compositions and/or combinations may
contain emulsifying agents to disperse uniformly throughout the
composition and/or combination an active ingredient or other
excipient that is not soluble in the liquid carrier. Emulsifying
agents that may be useful in liquid compositions and/or
combinations of the present invention include, for example,
gelatin, egg yolk, casein, cholesterol, acacia, tragacanth,
chondrus, pectin, methyl cellulose, carbomer, cetostearyl alcohol,
and cetyl alcohol.
[0118] Liquid pharmaceutical compositions and/or combinations may
also contain a viscosity enhancing agent to improve the mouth-feel
of the product and/or coat the lining of the gastrointestinal
tract. Such agents include acacia, alginic acid bentonite,
carbomer, carboxymethylcellulose calcium or sodium, cetostearyl
alcohol, methyl cellulose, ethylcellulose, gelatin guar gum,
hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl
methyl cellulose, maltodextrin, polyvinyl alcohol, povidone,
propylene carbonate, propylene glycol alginate, sodium alginate,
sodium starch glycolate, starch tragacanth, and xanthan gum.
[0119] Sweetening agents such as aspartame, lactose, sorbitol,
saccharin, sodium saccharin, sucrose, aspartame, fructose,
mannitol, and invert sugar may be added to improve the taste.
[0120] Preservatives and chelating agents such as alcohol, sodium
benzoate, butylated hydroxyl toluene, butylated hydroxyanisole, and
ethylenediamine tetraacetic acid may be added at levels safe for
ingestion to improve storage stability.
[0121] A liquid composition and/or combination may also contain a
buffer such as guconic acid, lactic acid, citric acid or acetic
acid, sodium guconate, sodium lactate, sodium citrate, or sodium
acetate. Selection of excipients and the amounts used may be
readily determined by the formulation scientist based upon
experience and consideration of standard procedures and reference
works in the field.
[0122] The solid compositions and/or combination of the present
invention include powders, granulates, aggregates and compacted
compositions and/or combinations. The dosages include dosages
suitable for oral, buccal, rectal, parenteral (including
subcutaneous, intramuscular, and intravenous), inhalant and
ophthalmic administration. Although the most suitable
administration in any given case will depend on the nature and
severity of the condition being treated, the most preferred route
of the present invention is oral. The dosages may be conveniently
presented in unit dosage form and prepared by any of the methods
well-known in the pharmaceutical arts.
[0123] Dosage forms include solid dosage forms like tablets,
powders, capsules, suppositories, sachets, troches and lozenges, as
well as liquid syrups, suspensions, aerosols and elixirs.
[0124] The dosage form of the present invention may be a capsule
containing the composition and/or combination, preferably a
powdered or granulated solid composition and/or combination of the
invention, within either a hard or soft shell. The shell may be
made from gelatin and optionally contain a plasticizer such as
glycerin and sorbitol, and an opacifying agent or colorant.
[0125] A composition and/or combination for tableting or capsule
filling may be prepared by wet granulation. In wet granulation,
some or all of the active ingredients and excipients in powder form
are blended and then further mixed in the presence of a liquid,
typically water that causes the powders to clump into granules. The
granulate is screened and/or milled, dried and then screened and/or
milled to the desired particle size. The granulate may be tableted,
or other excipients may be added prior to tableting, such as a
glidant and/or a lubricant.
[0126] A tableting composition and/or combination may be prepared
conventionally by dry blending. For example, the blended
composition and/or combination of the actives and excipients may be
compacted into a slug or a sheet and then comminuted into compacted
granules. The compacted granules may subsequently be compressed
into a tablet.
[0127] As an alternative to dry granulation, a blended composition
and/or combination may be compressed directly into a compacted
dosage form using direct compression techniques. Direct compression
produces a more uniform tablet without granules. Excipients that
are particularly well suited for direct compression tableting
include microcrystalline cellulose, spray dried lactose, dicalcium
phosphate dihydrate and colloidal silica. The proper use of these
and other excipients in direct compression tableting is known to
those in the art with experience and skill in particular
formulation challenges of direct compression tableting.
[0128] A capsule filling of the present invention may comprise any
of the aforementioned blends and granulates that were described
with reference to tableting; however, they are not subjected to a
final tableting step.
[0129] The active ingredient and excipients may be formulated into
compositions and/or combinations and dosage forms according to
methods known in the art.
[0130] In one embodiment, a dosage form may be provided as a kit
comprising COMPOUND I, or a pharmaceutically acceptable salt, free
base, hydrate, ester, solvate and/or prodrug thereof and
pharmaceutically acceptable excipients and carriers as separate
components. In one embodiment, a dosage form may be provided as a
kit comprising COMPOUND I, or a pharmaceutically acceptable salt,
free base, hydrate, ester, solvate and/or prodrug thereof, at least
one additional therapeutically active agent, and pharmaceutically
acceptable excipients and carriers as separate components. In some
embodiments, the dosage form kit allow physicians and patients to
formulate an oral solution or injection solution prior to use by
dissolving, suspending, or mixing COMPOUND I, or a pharmaceutically
acceptable salt, free base, hydrate, ester, solvate and/or prodrug
thereof with pharmaceutically acceptable excipients and carriers.
In one embodiment, a dosage form kit which provides COMPOUND I, or
a pharmaceutically acceptable salt, free base, hydrate, ester,
solvate and/or prodrug thereof which has improved stability when
compared to pre-formulated formulations of COMPOUND I, or a
pharmaceutically acceptable salt, free base, hydrate, ester,
solvate and/or prodrug thereof.
[0131] In one embodiment, pharmaceutical formulations or
compositions and/or combinations of the present invention contain
25-100% or 50-100% by weight of COMPOUND I, or a pharmaceutically
acceptable salt, free base, hydrate, ester, solvate and/or prodrug
thereof, as described herein, in the formulation or composition
and/or combination.
[0132] In another embodiment, the methods of the present invention
include administering a therapeutically effective amount of
Compound I or a pharmaceutically acceptable salt, free base,
hydrate, complex, or chelate (including metal chelates, such as
iron, zinc and others) thereof in the pharmaceutical formulations
or compositions and/or combinations described above. In a specific
embodiment, the methods are for preventing, reducing or treating
cancer in a subject. In another embodiment, the methods are for
killing cancer cells. In another embodiment, the methods are for
inducing cell cycle arrest in cancer cells.
[0133] The following examples further illustrate the present
invention but should not be construed as in any way limiting its
scope.
EXAMPLES
Example 1
Materials and Methods for Examples 2-7
[0134] Drugs and Reagents
[0135] COMPOUND I and deuterated COMPOUND I (COMPOUND I-d6) were
provided by APTOSE Biosciences (San Diego, Calif.).
Detergent-compatible protein assay kit, DC.TM. Protein Assay was
purchased from BioRad Laboratories, Inc. (Hercules, Calif.). The
CellTiter 96.RTM. Aqueous One Solution Cell Proliferation Assay
(MTS) was were purchased from Promega (Madison, Wis.). PARP, MCL-1,
BAD, BIK, Na.sup.+/K.sup.+ ATPase antibodies were from Cell
Signaling Technology, Inc. (Danvers, Mass.). pSer139 H2AX and ATM
antibodies were purchased from Abcam (Cambridge, UK). ABCG2
antibody was obtained from KAMIYA Biomedical (Tukwila, Wash.).
Ko143 and was pSer1981-ATM antibody obtained from Millipore Sigma
(St. Louis, Mo.). Olaparib was purchased from Selleckchem (Houston,
Tex.). Carboplatin and topotecan were obtained from UCSD Moores
Cancer Center Pharmacy.
[0136] Cell Types and Culture
[0137] The human Burkitt lymphoma cell line Raji was obtained from
the American Type Tissue Culture Collection and cultured in RPMI
1640 medium (ATCC) supplemented with 10% fetal bovine serum (ATCC,)
at 37.degree. C., 5% CO.sub.2. The COMPOUND I-resistant Raji
(Raji/COMPOUND IR) cell line was generated by exposure to
progressively higher concentrations of COMPOUND I over a period of
6 months. CAOV3 cells were obtained from ATCC and cultured in
complete DMEM supplemented with 10% fetal bovine serum. MCF7 vector
controlled and BRCA1 shRNA subclones were obtained from Dr. Simon
Powell (Memorial Sloan-Kettering Cancer Center) and cultured in
EMBM with 10% fetal bovine serum. MCF10A and hTERT-IMEC clones were
obtained from Dr. Ben Ho Park (Johns Hopkins University). HCT116
BRCA2.sup.+/+ cells and BRCA2.sup.-/- cells were obtained from Dr.
Samuel Aparicio (British Columbia Cancer Research Centre). PEO1 and
PEO4 cells were obtained from Dr. Sharon Cantor (University of
Massachusetts) and these cell lines were cultured under the same
conditions as previously published. Sakai et al., Functional
restoration of BRCA2 protein by secondary BRCA2 mutations in
BRCA2-mutated ovarian carcinoma, Cancer Res. 2009; 69 (16):6381-6;
Konishi et al., Mutation of a single allele of the cancer
susceptibility gene BRCA1 leads to genomic instability in human
breast epithelial cells, Proc. Natl. Acad. Sci. 2011; 108
(43):17773-8; Xu et al., CX-5461 is a DNA G-quadruplex stabilizer
with selective lethality in BRCA1/2 deficient tumours, Nature
Communications 2017; 8:14432, all of which are hereby incorporated
by reference.
[0138] Cytotoxicity Study
[0139] Cells were plated and treated with the indicated drugs in
96-well plates for 5 days. Cell viability was measured using
CellTiter 96 AQ.sub.ueous one solution (MTS) cell proliferation
assay purchased from Promega, and IC.sub.50 values were calculated
using GraphPad Prism 6 Software.
[0140] Biotinylation and Immunoblotting Procedure
[0141] To quantify ABCG2 expression, cells were
surface-biotinylated with EZ-LINK sulfo-NHS-SS-biotin (Thermo
Scientific, Pittsburg, Pa.) and subjected to Western blot analysis
as previously reported and subjected to western blot analysis. Tsai
C Y, Liebig J K, Tsigelny I F, Howell S B, The copper transporter 1
(CTR1) is required to maintain the stability of copper transporter
2 (CTR2). Metallomics 2015; 7:1477-87, which is hereby incorporated
by reference.
[0142] RNA-seq and qRT-PCR
[0143] Total cellular RNA was isolated using the RNeasy mini kit
(QIAGEN, Valencia, Calif.) from three independent samples for each
experiment. RNA-seq samples were submitted to the IGM Genomics
Center, University of California, San Diego, La Jolla, Calif.
(http://igm.ucsd.edu/genomics/) for library generation and
validation using Agilent Bioanalyzer. Sequencing was performed on
Illumina Sequencer HiSeq4000. Bioinformatic Analysis was conducted
by OHSU. The forward and reverse primers used for confirmation of
ABCG2 over-expression were: 5'-TTA-GGA-TTG-AAG-CCA-AAG-G-3' (SEQ ID
NO. 1) and 5'-TAG-GCA-ATT-GTG-AGG-AAA-ATA-3', (SEQ ID NO. 2)
respectively.
[0144] Cellular Pharmacology of COMPOUND I
[0145] Cells exposed to COMPOUND I or Fe(COMPOUND I).sub.3 were
homogenized in acetonitrile containing 5 ng of a deuterated
COMPOUND I standard. Samples were analyzed at the UCSD Molecular
Mass Spectrometry Facility employing an Agilent 1260 liquid
chromatograph (LC) system coupled with a Thermo LCQdeca mass
spectrometer using positive ion mode electrospray ionization (ESI)
as the ion source. The ESI ion source voltage was set at 5 kV, with
sheath gas flow rate of 80 units, auxiliary gas flow rate of 20
units, and capillary temperature of 250.degree. C., respectively. A
Phenomenex Kinetex Biphenyl column (ID 2.1 mm.times.length 50 mm,
particle size 2.6 .mu.m) was utilized for LC separation using water
with 0.1% formic acid as the mobile phase A and acetonitrile with
0.1% formic acid as the mobile phase B. The LC flow rate was set at
0.30 mL/min. The LC gradient increased from 5% mobile phase B to
95% mobile phase B in 10 minutes, held at 95% B for 2 minutes,
returned to 5% B in 1 minute, and then held at 5% B for 6 minutes.
Under positive ion mode ESI-MS/MS analysis, a major fragmental peak
of COMPOUND I was observed at m/z 353 from its molecular ion peak
at m/z 368 ([M+H]+) with a normalized collision energy of 45%, and
a major fragmental peak of COMPOUND I-d6 at m/z 359 from its
molecular ion peak at m/z 374 ([M+H]+) was observed with a
normalized collision energy of 45%. Selected reaction monitoring
(SRM) mode was used to acquire the m/z 353 and m/z 359 fragmental
peaks. The SRM peak area ratio (COMPOUND I/COMPOUND I-d6) related
to the amount of spiked COMPOUND I-d6 was used for the
quantification of COMPOUND I and Fe(COMPOUND I).sub.3 in the
samples. The same column, gradient and flow rate were used for
detection of Fe(COMPOUND I).sub.3 which was detected using an
Agilent 1100 HPLC and Orbitrap XL (Thermo) mass spectrometer
employing a Thermo IonMax ESI interface. The Fe(COMPOUND I).sub.3
eluted around 11.5 minutes with these conditions. A 10:1 flow split
was used for the eluent flow rate of 0.3 mL/min, so that
approximately 0.030 mL/min was introduced into the ESI after the
split. The ion source MS parameters were as follows: capillary
temperature 250.degree. C., sheath gas flow 20 units, positive
polarity, source voltage 5.0 kV, capillary voltage 22 V, and tube
lens 80 V. The Fourier transform MS (Orbitrap) parameters were:
FTMS AGC 1e6, FTMS microscans averaged 2, and FTMS full scan
maximum ion time 500 ms. The resolution parameter of 15,000 (peak
m/z divided by peak width given as full width at half maximum, at
400 m/z) was used. For the MS-MS CID spectra, a normalized
collision energy of 45% was used.
[0146] Synthesis and Characterization of Fe(COMPOUND I).sub.3
[0147] Five molar equivalents of ferrous ion as FeSO.sub.4 in a
concentrated water stock was added to COMPOUND I in ethanol which
produced a deep red precipitate that was subsequently dissolved in
DMSO and characterized by HPLC and mass spectrometry. Fe(COMPOUND
I).sub.3 was >95% pure and stable in the complete RPMI-1640
media for at least 5 days.
[0148] Comet Assay
[0149] Comet assay kits were purchased from Trevigen (Gaithersburg,
Md.) and neutral comet assay was performed according to the
manufacturer's instructions. Images were collected with a Keyence
Fluorescent Microscope (Keyence America, Itasca, Ill.) and
quantitated with OpenComet software.
[0150] Immunofluorescence Staining
[0151] Cells were harvested and washed with PBS twice, fixed in
Z-fix solution (buffered zinc formalin fixatives, Anatech, Inc,
Creek, Mich.) and permeabilized and blocked with 0.3% Triton X-100
in PBS containing 5% bovine serum albumin. The cells were then
incubated with .gamma.-H2AX antibody (1:250 dilution in 0.3% Triton
X-100 in PBS containing 1% bovine serum albumin) overnight followed
by three washes. Cells were incubated for 1 h with
fluorescent-conjugated secondary antibodies (1:1000 dilution)
followed by three washes. Slides were mounted with ProLong Gold
antifade reagent with 4',6-diamidino-2-phenylindole (DAPI) to stain
cell nuclei (Molecular Probes). Fluorescence was viewed with
Keyence Fluorescent Microscope using a 100.times. objective and
quantitated with Image J software (the National Institutes of
Health).
[0152] Statistical Analysis
[0153] All two-group comparisons utilized Student's t-test with the
assumption of unequal variance. Data are presented as mean .+-.SEM
of a minimum of three independent experiments.
Example 2
Cellular Pharmacology of COMPOUND I
[0154] Among the cell-types for which COMPOUND I exhibits potent
cytotoxicity lymphomas are of interest since most of the standard
chemotherapeutic agents used to treat this disease cause
myelosuppression which limits dose. For this reason, Raji Burkitt's
lymphoma cells were selected for study of the cellular pharmacology
of COMPOUND I. The intracellular accumulation of COMPOUND I in the
Raji cells was quantified by liquid chromatography tandem mass
spectroscopy (LC-MS/MS). COMPOUND I and its internal standard
COMPOUND I-d6 eluted from the LC column at .about.6.9 minutes with
sharp peak profiles. Raji cells accumulated COMPOUND I relatively
slowly with content approaching steady-state by 6 h (FIG. 6A).
[0155] Careful examination of the LC-MS/MS tracings identified a
minor peak that eluted from the LC column at .about.8.7 minutes
under the same reaction monitoring mode selected for the detection
of COMPOUND I. Using LC-HR-ESI-TOFMS (liquid chromatography high
resolution electrospray ionization time of flight mass
spectrometry) a peak was identified with an m/z 578.65 that also
eluted at .about.8.7 minutes. High resolution MS/MS analysis with
the Obritrap-MS demonstrated that this was a complex of COMPOUND I
with ferrous iron at 3 to 1 ratio (FIG. 1B). The structure of the
Fe(COMPOUND I).sub.3 ternary complex was characterized by
LC-MS-ESI. Two main features of the precursor ion mass spectrum
constrained the identification of the structure. The first was the
accurate mass measurement of the mass-to-charge ratio (m/z) of its
positive two charged ion by high resolution MS. The second feature
was the isotope distribution of the measured peak that showed that
the structure contained at least one atom of iron. In addition, the
MS-MS spectrum of the complex showed two fragment ions, one at 368
m/z that was identical to the free COMPOUND I, and an ion at 789
m/z that was consistent with iron and two remaining COMPOUND I
ligands. The calculated mass of the ternary complex, 578.6520 m/z,
was in very close agreement with the average m/z result observed on
each of several different days, 578.6519 m/z. The difference ratio
was -0.2 ppm, measured versus calculated. The inter-day standard
deviation was 0.0003 m/z, n=3, and the intra-day mass difference
ratio was consistently less than 1.0 ppm. This measure of agreement
is within the standard of 3 ppm, which is generally applied for
proof-of-structure for synthetic organic products. The presence of
iron was confirmed by the isotope pattern that is characteristic of
that element. Iron has 4 stable isotopes, .sup.54Fe, .sup.56Fe,
.sup.57Fe, and .sup.58Fe, with natural abundance of 5.85, 91.75,
2.12, and 0.28 percent, respectively. The MS peak that occurs due
to the .sup.54Fe isotope is distinctive because it does not
coincide with natural isotopes of carbon, hydrogen and nitrogen of
the COMPOUND I ligands. In the spectrum of the complex, its
calculated mass is 577.6542 m/z (.about.1 m/z less than the most
abundant isotope peak because the ion is charge plus two). The
average mass observed for this peak was 577.6545 m/z, with standard
deviation 0.0003 m/z. The difference ratio was 0.5 ppm, inter-day
with n=3. The intensity of the .sup.54Fe peak also consistently
measured about 6% of the ion abundance intensity of the main
.sup.56Fe peak, as expected from the natural abundance ratio. For
the measurement of the peak positions given above, the results were
recalibrated with respect to an internal standard of 391.2843 m/z,
an ion of diisooctylphthalate that is ubiquitous due to ambient
background.
[0156] It was discovered that the Fe(COMPOUND I).sub.3 complex
could be synthesized simply by adding FeSO.sub.4 to COMPOUND I in
ethanol. The purity of Fe(COMPOUND I).sub.3 was documented by HPLC
and the complex was found to be stable on storage. The IC.sub.50 of
Fe(COMPOUND I).sub.3 was 145.7.+-.0.5 nM, 1.5-fold less potent than
COMPOUND I presumably due to the difficulty of entering cells with
its positive doubly charged Fe ion (FIG. 1C). The relative uptake
of COMPOUND I and Fe(COMPOUND I).sub.3 was examined by treating
Raji cells with 0.5 .mu.M of each compound for 6 h and correcting
the intracellular concentrations on the basis of the ionization
efficiency of each molecule (FIG. 1D). COMPOUND I-treated cells
accumulated more intracellular Fe(COMPOUND I).sub.3 than the
Fe(COMPOUND I).sub.3-treated cells, consistent with the difference
in the IC.sub.50 of these two molecules. While the majority of
COMPOUND I was converted to Fe(COMPOUND I).sub.3 intracellularly in
the COMPOUND I-treated cells, Fe(COMPOUND I).sub.3 did not
dissociate intracellularly to produce detectable free COMPOUND I in
the Fe(COMPOUND I).sub.3-treated cells. Accordingly, it is believed
that Fe(COMPOUND I).sub.3 is the dominant active intracellular form
of COMPOUND I.
Example 3
COMPOUND I Causes DNA Damage
[0157] The structure of COMPOUND I is similar to drugs that bind to
quadruplex structures in DNA which results in strand breaks; this
led to the investigation of whether COMPOUND I caused damage to
DNA. The parental Raji cells were treated with 0.5 .mu.M COMPOUND I
for increasing periods of time and induction of DNA damage was
assessed by accumulation of the phosphorylated forms of ATM and
.gamma.H2AX measured by Western blot analysis. FIG. 2A shows that
COMPOUND I produced a clear increase in phosphorylated ATM and
.gamma.H2AX starting at 6 h in Raji cells and that this increased
with duration of drug exposure up to 24 h. Cleavage of PARP was
detected starting at 8 h indicating the induction of apoptosis.
Raji cells have very small nuclei making it difficult to quantify
the formation of .gamma.H2AX foci, so the human ovarian carcinoma
cell line CAOV3 was used for this purpose. FIG. 2B shows
representative images of .gamma.H2AX foci formation in the CAOV3
cells exposed to DMSO or 1 .mu.M COMPOUND I for 24 h. FIG. 2C shows
that an increase in the number of foci was detectable at 1 h and
that the number of foci increased more markedly after 8 h. Evidence
of DNA damage was further strengthened by the results of the
neutral comet assay which mainly detects DNA double strand breaks
(FIG. 2D). Although there was no increase in tail DNA when cells
were treated with 0.5 .mu.M COMPOUND I for 6 h compared to the DMSO
treatment, there was significantly more DNA in the comet tails when
cells were treated with COMPOUND I for 6 h and then incubated in
drug free media for 18 h (pulse-chase). These results provide
strong evidence that COMPOUND I generates DNA damage and produces
accumulation of DNA strand breaks capable of triggering
apoptosis.
Example 4
BRCA1/2 Deficient Cells are Hypersensitive to COMPOUND I
[0158] The finding that COMPOUND I produced DNA damage led to the
investigation of whether cells deficient in homologous
recombination were hypersensitive to this drug. The hypothesis that
there would be synthetic lethality between COMPOUND I and BRCA1
deficiency using isogenic pairs of BRCA1-proficient and -deficient
human cell lines was tested. Two independent MCF10A subclones, each
containing a heterozygous knockin of a 2-bp deletion in BRCA1 that
resulted in a premature termination codon (BRCA1-het #1 and #2),
were found to be more sensitive to olaparib than clones that
underwent random integration of the targeting vector within their
genomes (control) confirming the loss of BRCA1 function in the two
knockin clones (FIG. 3A, left). These two knockin clones were even
more hypersensitive to COMPOUND I (FIG. 3A, right). The effect of
impaired BRCA1 function was confirmed in a clone containing the
same 2-bp knock-in derived from the hTERT-IMEC cell line, when it
too was found to be hypersensitive to both olaparib and COMPOUND I
(FIG. 3B). The conclusion that BRCA1-deficient cells are
hypersensitive to COMPOUND I was further supported by the results
obtained in MCF7 E7 cells in which BRCA1 expression is stably
knocked down by the expression of an shRNAi. As shown in FIG. 3C,
the E7 clone has a similar degree of hypersensitivity towards
olaparib and COMPOUND I. These results in three independent
isogenic pairs of BRCA1 competent and BRCA1 incompetent cells
indicate that repair of the DNA damage produced by COMPOUND I is in
part dependent on homologous recombination and/or other DNA repair
pathways in which BRCA1 functions. Whether BRCA2-deficient cells
are more sensitive to COMPOUND I was tested using the
BRCA2-proficient and BRCA2-deficient ovarian cancer cell lines,
PEO4 and PEO1, respectively. PEO1 is BRCA2-deficient and sensitive
to cisplatin and a poly(ADP-ribose) polymerase inhibitor AG14361.
PEO4 was derived from ascites at the time of relapse with cisplatin
resistance and contains a secondary mutation that restores BRCA2
function. Restoration of BRCA2 function increased resistance to
both olaparib (FIG. 3D, left), and COMPOUND I (FIG. 3D, right).
Similar results were obtained using the BRCA2-proficient HCT116
cells and two BRCA2.sup.-/- subclones, B18 and B46 (FIG. 3E). Thus,
loss of either BRCA1 or BRCA2 function renders malignant cells
hypersensitive to COMPOUND I.
Example 5
Selection for Acquired Drug Resistance
[0159] In order to delineate which effects of COMPOUND I are most
closely linked to sensitivity for this drug, a subline of the Raji
Burkitt's lymphoma cell line that had acquired resistance
(Raji/COMPOUND IR) as a result of repeated exposure to
progressively higher concentrations of COMPOUND I over a period of
6 months was developed. Resistance evolved slowly and progressively
without an abrupt change at any point during the selection process.
The IC.sub.50 of COMPOUND I for the parental Raji cells was
91.9.+-.22.3 nM when tested using an assay that quantified growth
rate during a 120 h exposure to drug. This is in the same range as
has been reported for freshly isolated AML blasts and CLL cells.
Zhang et al., "Inhibition of c-Myc by ATPO-COMPOUND I as an
Innovative Therapeutic Approach to Induce Cell Cycle Arrest and
Apoptosis in Acute Myeloid Leukemia [abstract]," Blood 2016;
128:1716; Kurtz et al., "Broad Activity of COMPOUND I in AML and
other Hematologic Malignancies Correlates with KFL4 Expression
Level [abstract]," Blood 2015; 126:1358, both of which are hereby
incorporated by reference. The Raji/COMPOUND IR cells were
16.7.+-.3.9-fold resistant to COMPOUND I (IC.sub.50: 1387.7.+-.98.5
nM). The level of resistance remained stable for at least 3 months
during culture in drug-free media (FIG. 4A). Raji/COMPOUND IR cells
grew slightly faster than the parental cells but the difference was
not statistically significant. At a concentration that induced
apoptosis in the Raji sensitive cells, COMPOUND I failed to trigger
apoptosis in the Raji/COMPOUND IR cells. When the sensitive cells
were treated with 0.5 .mu.M COMPOUND I for 24 h, the pro-apoptotic
proteins BIK and BAD increased by 47.5.+-.16.8% and 2.1.+-.0.25
-fold, respectively (p<0.05, n=3) and the anti-apoptotic protein
MCL-1 decreased by 38.1.+-.2.3% (p<0.001, n=3) compared to the
DMSO control. None of these changes were detected in the
Raji/COMPOUND IR cells subjected to the same exposure (FIG.
4B).
Example 6
Mechanism of Drug Resistance
[0160] Resistance in the Raji/COMPOUND IR cells may be due to
alterations in influx or efflux, intracellular detoxification or a
change in the primary target of the drug. The intracellular
accumulation of both native COMPOUND I and the Fe(COMPOUND I).sub.3
in Raji and Raji/COMPOUND IR cells incubated with either native
COMPOUND I or the Fe(COMPOUND I).sub.3 complex was monitored. The
rate of accumulation of both forms of the drug was severely reduced
in the Raji/COMPOUND IR cells exposed to COMPOUND I (FIG. 6A and
Table 1).
TABLE-US-00001 TABLE 1 Rate of efflux of COMPOUND I and Fe(COMPOUND
I).sub.3 from Raji and Raji/COMPOUND IR cells over first 2 hours
(log fmole/10{circumflex over ( )}7 cells/h). Cell line
Raji/COMPOUND Raji IR COMPOUND I -0.19 .+-. 0.025* -0.26 .+-.
0.025* (-0.24~-0.13).sup. (-0.32~-0.21).sup. Fe (COMPOUND -0.22
.+-. 0.044* -0.17 .+-. 0.063* I).sub.3 (-0.32~-0.13).sup. (-0.32 to
-0.014).sup. *Mean .+-. SEM, n = 6 .sup. 95% Confidence
Intervals
The same was true to lesser extent when the cells were incubated
with the Fe(COMPOUND I).sub.3 complex (FIG. 6B). In contrast, there
was no apparent difference in the efflux over the first 2 h of
either ATPO-COMPOUND I or Fe(COMPOUND I).sub.3 following loading of
the cells with either form of the drug. These results indicate that
resistance to COMPOUND I in Raji cells is associated with impaired
accumulation of both forms of the drug. A more detailed measurement
of drug accumulation at 6 h confirmed that the accumulation of both
forms of the drug was markedly reduced when the Raji/COMPOUND IR
cells were incubated with COMPOUND I; however, the level of the
Fe(COMPOUND I).sub.3 complex still exceeded that of the native drug
(FIG. 4C). Only when the Raji/COMPOUND IR cells were treated with
at least 3 times as much COMPOUND I did the intracellular content
of Fe(COMPOUND I).sub.3 finally reach a level similar to that in
the sensitive cells (FIG. 4D). Treatment of the Raji/COMPOUND IR
cells with 0.5 .mu.M COMPOUND I for 24 h produced no increase in
phospho-ATM or phospho-.gamma.H2AX, and no detectable PARP cleavage
(FIG. 7) consistent with substantially less intracellular COMPOUND
I and Fe(COMPOUND I).sub.3 in the resistant cells.
[0161] To obtain further insight into the resistance mechanism,
RNA-seq analysis was carried out on three independent samples of
both the sensitive Raji and resistant Raji/COMPOUND IR cells. A
gene-level differential expression analysis was performed by
removing all genes with less than 50 reads across all 6 samples as
genes with only low level expression can cause irregularities in
differential expression analysis. Genes were considered to be
differentially expressed if their adjusted p-value was less than
the 0.05 level and their fold change was >2 in either direction.
Among the 13,791 evaluable genes there were 1,012 that were
significantly up-regulated in the Raji/COMPOUND IR cells and 704
genes that were significantly down regulated. The ATP-binding
cassette sub-family member ABCG2 was the most up-regulated gene
with more than a thousand-fold increase in transcript level (Table
2).
TABLE-US-00002 TABLE 2 Rank order of genes up-regulated in
Raji/COMPOUND IR cells. Gene ID Gene name Fold increase Adjusted p
value ENSG00000118777 ABCG2 1127.3 2.24E-05 ENSG00000114200 BCHE
173.8 3.24E-05 ENSG00000142149 HUNK 52.9 8.59E-04 ENSG00000060709
RIMBP2 46.6 3.99E-04 ENSG00000165695 AK8 42.5 5.74E-04
ENSG00000234323 RP11- 41.4 3.16E-04 308N19.1 ENSG00000261690
AC009133.12 39.3 1.60E-03 ENSG00000161570 CCL5 37.3 5.74E-05
ENSG00000168824 NSG1 33.0 9.84E-04 ENSG00000154864 PIEZO2 31.6
3.88E-04
Although several other multidrug resistance ABC transporters were
also up-regulated in Raji/COMPOUND IR, the increase in ABCG2
transcripts was the most prominent (Table 3). The marked
up-regulation of ABCG2 in the Raji/COMPOUND IR cells was confirmed
by qRT-PCR and Western blot analysis (FIGS. 5A and B).
TABLE-US-00003 TABLE 3 ABC transporter family member genes
up-regulated in Raji/COMPOUND IR cells. Gene ID Gene name Fold
increase Adjusted p value ENSG00000118777 ABCG2 1127.3 2.24E-05
ENSG00000160179 ABCG1 0.9 8.56E-01 ENSG00000085563 ABCB1 4.8
8.24E-04 (MDR1) ENSG00000103222 ABCC1 1.5 1.25E-02 (MRP1)
ENSG00000023839 ABCC2 3.2 6.09E-03 (MRP2)
[0162] Ko143 is a specific ABCG2 inhibitor with more than 200-fold
selectivity relative to its ability to inhibit the P-gp or MRP-1
transporters. Ko143 itself was not toxic to Raji or Raji/COMPOUND
IR cells at concentrations up to 300 nM (FIG. 5C). To test the
hypothesis that COMPOUND I is a substrate for ABCG2, the ability of
Ko143 to reverse the resistance of the Raji/COMPOUND IR cells was
evaluated. The data in Table 4 and FIG. 5D show that concurrent
treatment with Ko143 significantly reversed COMPOUND I resistance
in the Raji/COMPOUND IR cells.
TABLE-US-00004 TABLE 4 Effect of ABCG2 inhibitor on resistance to
COMPOUND I COMPOUND I COMPOUND I + COMPOUND I + Alone 5 nM Ko143 50
nM Ko143 IC.sub.50 IC.sub.50 IC.sub.50 Cell Line (nM).sup.#
RR.sup..sctn. (nM) RR.sup..sctn. (nM) RR.sup..sctn. Raji/COMPOUND
1387 .+-. 94 16.7 .+-. 3.9.sup.b 853 .+-. 44 10.9 .+-. 1.9.sup.c
200.6 .+-. 20.7 2.5 .+-. 0.7.sup.a IR Raji .sup. 105 .+-. 2.4 --
98.3 .+-. 0.8 -- 103.1 .+-. 2.9 -- .sup.#Mean .+-. SEM
.sup..sctn.Relative resistance .sup.ap < 0.05; .sup.bp <
0.01; .sup.cp < 0.001
[0163] To provide further evidence of augmented ABCG2 function, the
resistant cells were tested for cross-resistance to topotecan, a
well-documented ABCG2 substrate. The Raji/COMPOUND IR cells were
found to be 3-fold cross-resistant to topotecan and treatment with
Ko143 reversed this resistance completely (FIG. 5E). Intriguingly,
Raji/COMPOUND IR was also significantly cross-resistant to
carboplatin even though carboplatin is not thought to be an ABCG2
substrate; treatment with Ko143 did not reduce the carboplatin
IC.sub.50 in the Raji/COMPOUND IR cells (FIG. 5F). Surprisingly,
Raji/COMPOUND IR cells were found to be hypersensitive to
etoposide, an ABCG2 substrate and potent double strand break
inducer. GO and pathway analysis from the RNA-seq data revealed
that the DNA repair pathways were downregulated in Raji/COMPOUND IR
which partially explained the hypersensitivity to etoposide.
Example 7
COMPOUND I Interaction With G-Quadruplex DNA is Linked to
Inhibition of c-MYC
[0164] Current mechanistic studies demonstrated that COMPOUND I
modulates c-MYC at the transcriptional level by decreasing
acetylated H3K27 at its promoters and additionally by destabilizing
c-MYC mRNA. In addition, differential gene expression analysis of
RNA-seq and reverse phase protein array (RPPA) data highlighted a
role for c-MYC in the mechanism of COMPOUND I (GO terms--Down
regulated by c-MYC p-value 6E-26, Gene promoters bound by c-MYC
p-value 4.2 E-10, ChIP targets of c-MYC p-value 3.3E-8).
Furthermore, from the RPPA data an increase in p-Chk1, p-Chk2,
.gamma.H2Ax, and total p53 and E2F1 was observed, all of which are
indicative of activation of DNA damage response pathways. This was
accompanied by elevated levels of XBP1, GRP78, and p-p38 that point
towards cellular stress response signaling (GO term Regulation of
Cell Stress, p-value 1.89E-8).
[0165] Although COMPOUND I may participate in multiple mechanistic
events, the effect of COMPOUND I on c-MYC expression, cell cycle
arrest and DNA damage, as well as synthetic lethality in cells with
compromised DNA repair mechanisms, can be explained by the action
of the Fe(COMPOUND I).sub.3 complex on G-quadruplex DNA motifs.
Example 8
Materials and Methods for Examples 9-16
[0166] Cells and Compounds
[0167] EOL-1, GRANTA-519, Jeko-1, Jurkat, Molm-13, NOMO-1, SKM-1,
and SU-DHL-6 were obtained from Leibniz-Institut DSMZ. HL-60, KG-1,
Mino, MV4-11, Raji, and THP-1 were obtained from ATCC. HEL92.1.7
were obtained from the European Collection of Authenticated Cell
Cultures and Ramos cells were a gift from Dr. M. Andreeff (MD
Anderson Cancer Center, Houston, Tex.). All cells were cultured in
complete media as per the manufacturer's instructions. Early
passage cells were collected and frozen within 1 month of receipt
from the manufacturer. All experiments were performed on early
passage cells within 6 weeks of thawing. MycoScope Mycoplasma
Detection Kit (Genlantis catalog #MY01050) was used to screen for
potential contamination every 6 months. Peripheral blood
mononuclear cells (PBMC) were isolated from fresh healthy donor
blood using Ficoll-Paque PLUS (GE Healthcare, catalog #17-1440-02).
For synthesis of COMPOUND I free base, 10-phenanthroline-5,6-dione
(1.2 equivalents), acetic acid (22 volumes),
2-methyl-5-fluoroindole-3-carboxaldehyde (1.0 equivalents), and
ammonium acetate (15 equivalents) were reacted under medium
agitation while heated at 95.+-.5.degree. C. for 3 to 7 hours. The
reaction was cooled to between 20.degree. C. and 25.degree. C.,
filtered, rinsed with acetic acid and ethanol, and dried with
N.sub.2 purge, followed by a wash with 2:1 ethanol:water at
65.degree. C. for 4 hours, cooling to 20.degree. C. to 25.degree.
C., filtration, rinsing with 2:1 ethanol:water and EtOAc, and then
dried with N.sub.2 purge. The purity by HPLC was 99.5%, and the
structural identity was confirmed by FT-IR, .sup.1HNMR, .sup.13C
NMR, and LC/MS. For Fe(COMPOUND I).sub.3 synthesis, five molar
equivalents of ferrous ion FeSO.sub.4 in water was added to
COMPOUND I dissolved in ethanol. The deep red precipitate produced,
Fe(COMPOUND I).sub.3, was collected and dissolved in DMSO and
characterized by HPLC and mass spectrometry as >95% pure.
CX-5461 (7) was purchased from MedChem Express (catalog
#HY-13323).
[0168] Cytotoxicity Study
[0169] Cells were plated and treated with vehicle DMSO or COMPOUND
I (10 concentrations) in 96-well plates for 5 days at 37.degree. C.
and 5% CO.sub.2. Cell viability was measured using CellTiter 96
AQ.sub.ueous one solution (MTS) cell proliferation assay (Promega,
catalog #G3581), and IC.sub.50 values were calculated using
GraphPad Prism 7 software.
[0170] Uptake and Efflux Assay
[0171] Cells exposed to COMPOUND I were homogenized in acetonitrile
containing 5 ng of deuterated COMPOUND I standard. Samples were
analyzed at the UCSD Molecular Mass Spectrometry Facility employing
an Agilent 1260 liquid chromatograph (LC) system coupled with a
Thermo LCQdeca mass spectrometer using positive ion mode
electrospray ionization as the ion source.
[0172] qRT-PCR
[0173] Cells were treated with vehicle or COMPOUND I at various
concentrations for 24 hours or at a single concentration for 1, 3,
6, 12, and 24 hours before harvesting. Cells were lysed by
QiaShredder columns (QIAGEN, catalog #79656), total RNA was
isolated using QIAGEN RNeasy Plus Mini Kit (catalog #74134), and
cDNA was synthesized utilizing Transcriptor Universal cDNA master
mix (Roche, catalog #05893151001) and then used for qRT-PCR
analysis using FastStart essential DNA probes master mix (Roche,
catalog #06402682001) and Roche LightCycler96. Primer probe pairs
were purchased from IDT (Table 5). Expression was calculated as
fold change over vehicle treated samples after normalizing to GAPDH
(2.sub.t.sup..DELTA..DELTA.C).
TABLE-US-00005 TABLE 5 IDT primer probe pairs Gene IDT assay name
GAPDH Hs.PT.58.40035104 CDKN1A (p21) Hs.PT.58.40874346 MYC
Hs.PT.58.26770695
[0174] Western Blotting
[0175] Cells were treated as described above. Whole-cell lysates
were prepared, separated by SDS-PAGE, and transferred to
nitrocellulose membranes. Detection antibodies used are listed in
Table 6. Densitometry was performed using ImageJ or Image Studio
Lite Version5.2 software and normalized to the density of
GAPDH.
TABLE-US-00006 TABLE 6 Antibodies Antibodies Cat# Company
CCND3/Cyclin D3 2936 Cell Signaling CDK4 12790 Cell Signaling PARP1
9532 Cell Signaling Total TP53 sc-126 Santa Cruz TP53 Phos-Ser15
2528 Cell Signaling TP53 acetyl K382 2525 Cell Signaling
.gamma.H2AX 9718 Cell Signaling CHEK1 phos-Ser345 2348 Cell
Signaling CHEK1 2360 Cell Signaling MAPK14/p38 phos- 4511 Cell
Signaling Thr180/Tyr182 MAPK14/p38 8690 Cell Signaling MAPK8/JNK
phos- 4668 Cell Signaling Thr183/Tyr185 CDKN1A/p21 sc-397 Santa
Cruz GAPDH sc-365062 Santa Cruz MYC sc-40 Santa Cruz RIgG sc-2025
Santa Cruz 20 Rabbit HRP 170-8515 Biorad 20 Mouse HRP 170-6516
Biorad
[0176] Flow Cytometry for Apoptosis and Cell-Cycle Analysis
[0177] Cells were treated as described above. To determine
apoptosis, cells were stained with FITC-Annexin V and propidium
iodide (PI; BD Pharmingen, catalog #556570) and then analyzed on BD
Accuri C6 flow cytometer. To measure DNA synthesis and phases of
cell cycle, treated cells were stained with
5-ethynyl-2'-deoxyuridine (Edu) Alexa Fluor 488 (Thermo Fisher
Scientific, catalog #C10425) and PI (PI/RNase A staining solution,
BD Biosciences, catalog #550825). The dead cells were excluded from
analysis by using Live/Dead Fixable Far Red Dead Cell Stain Kit
(Thermo Fisher Scientific, catalog #L34973). Staining was performed
as per the manufacturers' instructions.
[0178] RNA Sequencing Analysis
[0179] Treated MV4-11 cells were subjected to total RNA extraction
(as for qRT-PCR analysis) and sequenced at the UCSD genomics core
facility. RNA was processed using Illumina TruSeq and single end
sequenced for 50-bp reads on the Illumina HiSeq4000. Data were
analyzed by the McWeeny lab at Oregon Health and Science University
(Portland, Oreg.). FASTQ files were assessed for read base
distribution and sequence representation using FASTQC
http://www.bioinformatics.babraham.ac.uk/projects/fastqc/. Reads
were aligned to HG37 using SubRead v1.5.0-pl keeping uniquely
mapped reads. Differential expression genes with less than 50 reads
(across all 4 samples) were discarded. Raw data and processed files
are available on GEO
(https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE111949)
accession number GSE111949.
[0180] Reverse-Phase Protein Array Analysis
[0181] MV4-11 cells were treated as for RNA sequencing (RNA-seq)
analysis and whole-cell extracts were prepared for Western
blotting. Samples were processed at MD Anderson Cancer Center
reverse-phase protein array (RPPA) core facility (details at
https://www.mdanderson.org/research/research-resources/core-facilities/fu-
nctional-proteomics-rppa-core/rppa-process.html). Protein
expression levels were averaged for 3 replicates and heatmaps were
drawn using GraphPad Prism 7.
[0182] Chromatin Immunoprecipitation Coupled With qPCR
[0183] MV4-11 cells were treated with vehicle DMSO or 500 nmol/L
COMPOUND I for 2, 6, or 24 hours and then crosslinked with 1%
formaldehyde. Chromatin was extracted by sonication and then
incubated with H3K27ac (Active Motif #39133) antibody overnight.
The antibody:DNA complexes were isolated with Protein G beads
(Invitrogen Dynabeads catalog #10004D) and analyzed by qPCR with
primers specific to the MYC promoter (Table 7). H3K27ac enrichment
was calculated as fold over input DNA control.
TABLE-US-00007 TABLE 7 ChIP Primers Location forward primer reverse
primer MYC Prom1 GAGCAGCAGCGAAAGGGAGA CAGCCGAGCACTCTAGCTCT (SEQ
(SEQ ID NO. 3) ID NO. 4) MYC Prom 2 CCGCATCCACGAAACTTTG
GGGTGTTGTAAGTTCCAGTGCAA (SEQ ID NO. 5) (SEQ ID NO. 6)
[0184] RNA Decay Assay
[0185] Cells were treated for 3 hours with vehicle DMSO or 500
nmol/L COMPOUND I followed by 1 .mu.mol/L actinomycin D. Aliquots
of cells were taken before and then every 10 minutes after
actinomycin D addition for RNA extraction and cDNA synthesis as for
qRT-PCR analysis. Levels of MYC and 28s rRNA were analyzed using
specific primers (Table 8) and MYC RNA expression was normalized to
28 s rRNA [2 (28 s C.sub.t value-MYC C.sub.t value)].
TABLE-US-00008 TABLE 8 Expression Primers Gene forward primer
reverse primer 28s RNA AGTAGCAAATATTCAAACGAGAACTTT
ACCCATGTTCAACTGCTGTTC (SEQ ID NO. 7) (SEQ ID NO. 8) MYC
CAGTAGAAATACGGCTGCAC (SEQ ID TTCGGGTAGTGGAAAACCAG NO. 9) (SEQ ID
NO. 10)
[0186] FRET Assay
[0187] FRET assay and data analysis was performed as described
previously and modified by using dual labeled (5' FAM-3' BHQ1)
single-stranded oligos. Melting temperature of each oligo was
assessed in the presence of vehicle DMSO or escalating
concentrations of COMPOUND I, Fe(COMPOUND I).sub.3, CX-5461, or
TMPyP4 using a Roche LightCycler 96 [at 37.degree. C. for 300
seconds followed by temperature increased in 3.degree. C. intervals
up to 91.degree. C. (25 steps) with 300-second total incubation
time at each temperature]. Drug and oligo reaction mixes were
analyzed immediately or incubated for 6 hours at room temperature
and then analyzed. Primer information is provided in Table 9.
Longer incubation time did not affect Fe(COMPOUND I).sub.3, TMPyP4,
or CX-5461 activity but enhanced COMPOUND I G4-binding ability.
COMPOUND I data are presented for 6-hour time point.
TABLE-US-00009 TABLE 9 G-quadruplex oligos G4 Location G4 oligo
sequence Telomere 5'(FAM)-GGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGG-
(BHQ1)3' (SEQ ID NO. 11) MYC 5'(FAM)-CCATGGGGAGGGTGGAGGGTGGGGAAGGT-
(BHQ1)3' (SEQ ID NO. 12) KIT
5'(FAM)-TTATAGGGAGGGCGCTGGGAGGAGGGAGGAGAC- (BHQ1)3' (SEQ ID NO. 13)
rRNA 5'(FAM)-AATAAGGGTGGCGGGGGGTAGAGGGGGGTAATA- (BHQ1)3' (SEQ ID
NO. 14) ds_DNA 5'(FAM)-TATAGCTATA[Sp~C18]TATAGCTAT-(BHQ1)3' (SEQ ID
NO. 15)
Example 9
COMPOUND I Induces Cytotoxicity, Upregulates p21, and Induces
G.sub.0-G.sub.1 Cell-Cycle Arrest in AML Cells
[0188] COMPOUND I inhibited proliferation in AML cell lines and
various forms of lymphoma cell lines with IC.sub.50 values ranging
from 57 nmol/L to 1.75 .mu.mol/L (FIG. 8; Table 10). The drug
showed only modest variation in potency as a function of duration
of exposure in MV4-11 cells with IC.sub.50 values of 0.47, 0.40,
and 0.24 .mu.mol/L for exposures of 48, 72, and 120 hours,
respectively. Previous studies reported upregulation of KLF4 and
CDKN1A expression as a potential mechanism of APT0-COMPOUND I
activity in tumors. Although COMPOUND I upregulates KLF4 expression
in 4 of 6 AML cell lines tested (FIG. 15A), CDKN1A (p21) expression
was induced in all AML cell lines in a concentration-dependent
manner (FIGS. 15B and 15C). The upregulation of CDKN1A mRNA
increased with duration of exposure in all AML cell lines tested
(FIGS. 15D and 15E). At later time points, CDKN1A expression began
to decrease, likely due to increasing cell death. Induction of
CDKN1A expression is typically associated with a subsequent
G.sub.0-G.sub.1, cell-cycle arrest, which was observed following
COMPOUND I treatment of solid tumor lines. Consistent with this, a
dose-dependent increase of cells in G.sub.0-G.sub.1 was observed
with concomitant reduction in the fraction of cells in the S- and
G.sub.2-M phases for all AML cell lines tested (FIGS. 9A-C, top).
In the case of MV4-11, all live cells had arrested in
G.sub.0-G.sub.1 after 24-hour exposure to 1 .mu.mol/L APT0-COMPOUND
I. CCND3 (Cyclin D3) and CDK4 are known to promote G.sub.1
cell-cycle progression, while CDKNIA serves to negatively regulate
this process. Western blot analysis of COMPOUND I-treated AML cells
revealed dose-dependent inhibition of both CDK4 and CCND3, albeit
to different degrees in each of the 3 AML lines (FIGS. 9A-C,
bottom; FIG. 16A).
TABLE-US-00010 TABLE 10 COMPOUND I IC.sub.50 values in leukemia and
lymphoma cell lines Disease IC.sub.50 (.mu.M) Type Cell Lines Mean
MCL Jeko-1 0.057 MCL GRANTA-519 0.082 Burkitt's Raji 0.1 AML
MOLM-13 0.14 MCL Mino 0.23 AML MV4-11 0.24 AML EOL-1 0.3 AML THP1
0.34 Burkitt's Ramos 0.35 AML HL-60 0.46 AML SKM-1 0.48 AML KG-1
0.51 DLBCL SUDHL-6 0.51 T-ALL Jurkat 0.52 AML Nomo-1 1.45 AML
HEL92.1.7 1.75
[0189] To correlate cell-cycle arrest with various pathway
perturbations, and to delineate the sequence of mechanistic events,
cell-cycle analyses were performed after treating cells with either
vehicle or COMPOUND I (IC.sub.50 concentration) for various times
up to 24 hours. There was no perturbation of cell-cycle phase
distribution in cells treated with vehicle alone. An increase in
the fraction of cells in G.sub.0-G.sub.1 was detected as early as 2
hours, and this fraction continued to increase in a time-dependent
manner throughout the 24-hour period of drug exposure (FIGS. 9D-F).
Western blot analysis showed a time-dependent decrease of CDK4 and
CCND3 protein levels that paralleled the G.sub.0-G.sub.1 arrest
(FIGS. 16B and 16C). These data establish that COMPOUND I produces
a time- and concentration-dependent G.sub.0-G.sub.1 arrest in AML
cells and suggest that this is mediated by established p21 and
cyclin-dependent kinase pathways.
Example 10
COMPOUND I Induces Apoptosis in AML Cell Lines
[0190] To investigate the mechanism by which COMPOUND I causes cell
death, MV4-11, EOL-1, and KG-1 AML cells were treated with or
without COMPOUND I and subjected to apoptotic marker detection by
flowcytometry and Western blotting. Cells were stained with PI and
Annexin V to distinguish between live (Annexin V and PI negative),
early apoptotic (Annexin V positive and PI negative), late
apoptotic (Annexin V and PI positive), and dead (Annexin V negative
and PI positive) cells. A concentration-dependent increase in
apoptotic cells was observed at 24 hours in all cell lines (FIG.
10A; FIG. 17A). The IC.sub.50 values based on Annexin V and PI
staining paralleled antiproliferative IC.sub.50 values. PARP
cleavage (c-PARP1) is a classic signal of apoptosis downstream of
both the intrinsic and extrinsic pathways. COMPOUND I induced
accumulation of c-PARP1 in a concentration- and time dependent
manner that paralleled apoptosis induction as measured by Annexin
V/PI staining (FIGS. 10B-C; FIG. 17B). For all 3 AML cell lines,
increases in apoptotic cells appeared between 3 and 6 hours after
exposure to COMPOUND I (FIG. 10D), which followed G.sub.0-G.sub.1
cell-cycle arrest observed at 2-hour exposure.
Example 11
COMPOUND I Pharmacodynamics
[0191] To gain further insight into the pathways exploited by
COMPOUND I to cause cell-cycle arrest and apoptosis, differential
expression analysis was performed at both the mRNA and protein
levels. MV4-11 cells were treated with either vehicle or 500 nmol/L
COMPOUND I for 6 hours, and then gene expression was analyzed by
RNA-seq. A total of 1,643 genes were found to be differentially
regulated upon COMPOUND I treatment (>2-fold change and
P<0.05) with 416 being upregulated and 1,227 being downregulated
(Table 11). The RNA-seq analysis detected a 2-fold increase in KLF4
and a 4.5-fold increase in CDKN1A expression, which is validated by
the qRT-PCR data with MV4-11 cells. The differentially regulated
genes were analyzed for enriched pathways or GO (Gene Ontology)
terms (FIG. 18A) utilizing the Broad Molecular Signatures database
(http://software.broadinstitute.org/gsea/msigdb/index.jsp). As
expected, apoptotic and cell-cycle pathways were enriched in the
differentially expressed gene set. Unexpectedly, gene expression
profiles after COMPOUND I treatment were also enriched in the DNA
damage response (DDR) and endoplasmic reticulum (ER)
stress/unfolded protein response pathways. In addition, upregulated
genes were enriched in TP53 pathways and genes downregulated by
MYC. Gene expression changes detected in MV4-11 cells raised the
possibility that COMPOUND I could cause apoptosis by inducing DNA
damage and activating cellular stress pathways, and/or by
inhibition of expression of the MYC oncogene.
TABLE-US-00011 TABLE 11 Differential expression genes from RNA-seq
and RPPA antibody/protein fold change p-value Histone-H3 5.08312399
5.45E-05 H2AX_pS140 4.19597407 0.000522 PAR 2.70749443 0.001439
Caspase-3 2.38247687 0.00075 p38_pT180_Y182 2.02214932 0.000782
DM-Histone-H3 2.00835777 0.000457 DM-K9-Histone-H3 2.00341212
0.002556 Caspase-7-cleaved 1.67071017 3.46E-05 Bad_pS112 1.55036509
0.002505 Hif-1-alpha 1.4823094 0.012889 Syk 1.43304918 0.031588
Chk1 1.43276703 0.034112 E2F1 1.42334521 0.021131 N-Ras 1.39693163
0.004314 Ubq-Histone-H2B 1.37487462 0.037092 XBP-1 1.36926109
0.011104 Cyclin-E1 1.36142448 0.0002 Chk2_pT68 1.31238742 0.000457
ADAR1 1.3084933 0.007035 ACC_pS79 1.3015573 0.000181 CD29
1.29122101 0.032909 BiP-GRP78 1.28988582 0.008195 p53 1.28815199
0.035039 Pdcd-1L1 1.26162272 0.039318 PAI-1 1.25745484 0.025156
Rb_pS807_S811 0.64862926 0.005124 S6_pS240_S244 0.64668982 0.002977
Ets-1 0.60342423 0.014415 Cyclin-B1 0.59444823 0.001083 PLK1
0.49462682 0.009377 NDRG1_pT346 0.4566332 5.11E-05
[0192] To examine the effect of COMPOUND I on protein expression,
MV4-11 cells were treated as above and analyzed by RPPA microarray
to quantify >300 total and post-translationally modified
proteins. Effects were observed on levels of both total and
post-translationally modified proteins (>1.25-fold and
P<0.05) with more proteins unregulated than downregulated (FIG.
18B; Table 11). Of note, there was an increase of cleaved
caspase-7, which is indicative of apoptosis. GO analysis of the
differentially expressed proteins was performed utilizing the Broad
Molecular Signatures database. Significant GO terms included cell
death and G.sub.1-S cell-cycle arrest pathways, a formal
description of G.sub.0-G.sub.1 arrest, and consistent with the
cell-cycle effects detected by flow cytometry and the RNA-seq
analyses (FIG. 18C). An increase in E2F1, TP53, .gamma.H2AX, CHEK1
phos-S296, and CHEK2 phos-T68 supported the concept that DNA damage
pathways are triggered by COMPOUND I treatment. In addition,
increases in XBP1, HSPA5, and MAPK14 (p38) phos-T180/182 were
observed, indicating ER or cellular stress (P=1.89E.sup.-0.8; ref
15). DDR pathways can also signal through the MAPK pathway
activating MAPK14 and MAPK8 (JNK), and cross-talk between the DDR
pathway and ER stress is a well-established phenomenon although it
is unclear which pathway represents the initiating event. A
significant portion of the differentially expressed proteins and
mRNAs are target genes of MYC oncoprotein, which is known to be an
integral part of both cell-cycle and apoptotic pathway regulation.
Collectively, these data suggested that regulation of the MYC
oncogene may play an early and key role in the mechanism of
COMPOUND I.
Example 12
COMPOUND I Concentration- and Time Dependently Downregulates MYC
mRNA and Protein Levels in AML Cells
[0193] MYC expression is implicated in the pathogenesis of a wide
range of cancers, including leukemia and lymphoma. A recent study
demonstrated that inhibition of MYC transcription leads to
apoptosis in cancer cells of hematologic origin, making MYC an
attractive therapeutic target. A review of our RNA-seq dataset
revealed that MYC was downregulated by COMPOUND I in MV4-11 cells
at 6 hours. It was also observed that an increased transcription of
genes negatively regulated by MYC in COMPOUND I-treated MV4-11
cells. COMPOUND I produced a concentration-dependent decrease in
both MYC mRNA and protein levels in all AML cell lines tested, and
the IC.sub.50 values for MYC inhibition paralleled the
antiproliferative IC.sub.50 values (FIGS. 11A and B). These changes
increased as a function of exposure time up to 24 hours in the
MV4-11, EOL-1, and KG-1 AML cells (FIG. 11C; FIGS. 19A and 19B).
The time course of MYC protein repression in MV4-11 cells
paralleled inhibition of MYC gene expression levels detected by
RNA-seq. All the tested AML cell lines had significantly higher
basal expression of MYC as compared with PBMCs from healthy donors
(FIG. 11D; FIG. 19C). Thus, COMPOUND I downregulates MYC at the
mRNA and protein level in all AML cell lines examined.
[0194] Regulation of MYC expression is a complex process that
involves MYC transcription, mRNA stability, and protein turnover.
ChIP-qPCR analysis for H3K27ac, a well-established marker of active
chromatin, was performed to assess transcriptional competency of
the MYC gene promoter after treatment with COMPOUND I (FIG. 19D). A
decrease in H3K27ac at the MYC promoter in MV4-11 cells was
observed as early as 2 hours and progressed over time, indicating
that modification of the MYC promoter and subsequent
transcriptional repression of the MYC gene is an early mediator of
the COMPOUND I mechanism of action (FIG. 19E). To determine whether
COMPOUND I affected MYC mRNA stability, an RNA decay assay was
performed on EOL-1 cells. There was a clear decrease in MYC mRNA
levels in the COMPOUND I pretreated cells versus vehicle (FIG.
19F), indicating that COMPOUND I can decrease the stability of MYC
mRNA. These data suggest that COMPOUND I regulates MYC by affecting
both transcription and mRNA stability.
Example 13
COMPOUND I Triggers DNA Damage and Cellular Stress Pathways
[0195] In addition to MYC, RNA and protein differential expression
analyses pointed to the involvement of TP53, DNA damage, and ER
stress in the mechanism of action of COMPOUND I. Validation of the
RPPA data that had demonstrated an increase in TP53 protein level
after COMPOUND I treatment in MV4-11 cells was sought. Exposure of
MV4-11 cells to 500 nmol/L COMPOUND I produced a significant
increase in TP53 levels at early time points (1, 3, and 6 hours),
followed by a return to baseline at 12 hours and a further
reduction at 24 hours, presumably due to extensive cell death at
this time point (FIG. 12A). The increase in total protein was
concomitant with an increase in phospho-Ser15 and acetyl-K382 (FIG.
12B). TP53 is phosphorylated at Ser15 and Ser20 in response to DNA
damage, which reduces MDM2 binding and proteasomal degradation of
p53. Furthermore, p53 is acetylated in response to cellular stress,
and this modification can further stabilize TP53 protein levels and
modulate binding activity. Activation of TP53 can trigger apoptosis
through upregulation of proapoptotic factors such as BBC3 (PUMA),
PMAIP1 (NOXA), and BAX. The RNA-seq dataset showed a 3.95-fold
increase in BBC3 and 1.38-fold increase in PMAIP1 in COMPOUND
I-treated MV4-11 cells. Involvement of DNA damage and cell stress
pathways was further interrogated in MV4-11 cells at early time
points after treatment with 500 nmol/L COMPOUND I. An increase in
phos-CHEK1 was detected at 1 hour after COMPOUND I addition with a
peak at approximately 4 hours, suggesting that DNA damage was an
early event (FIG. 12C). Following CHEK1 phosphorylation, there was
a robust increase in the DDR marker H2AX by 6 hours. A
concentration-dependent increase in H2AX was detected in all AML
lines tested, thereby adding further credence to the concept that
COMPOUND I triggers the DDR pathway (FIG. 12D). In addition, there
was an increase in both MAPK14 phos-T180 and MAPK8
phos-Thr183/pTyr185 at 4- to 6-hour treatment, which indicated
signaling through the DDR or ER stress pathways (FIG. 12C).
Overall, the data suggest that DNA damage induced by COMPOUND I is
an early event in the mechanism of COMPOUND I.
Example 14
Intracellular Pharmacokinetics of COMPOUND I
[0196] Measurement of the kinetics of uptake and efflux of COMPOUND
I in KG-1 AML cells determined by mass spectrometry indicated a
gradual approach to steady state and a rapid initial efflux, but a
very prolonged terminal efflux (FIGS. 20A and 20B). When KG-1 cells
were exposed to COMPOUND I for either 1 or 6 hours and then placed
in drug-free media, the efflux pattern consisted of a rapid phase
occurring during the first 30 minutes followed by a prolonged
terminal phase, such that significant amounts of COMPOUND I were
retained in KG-1 cells for longer than 24 hours. Consistent with
these data, cellular pharmacokinetic studies disclosed that
COMPOUND I is transformed intracellularly to a complex containing 1
atom of Fe and 3 molecules of COMPOUND I [Fe(COMPOUND I).sub.3]
(FIGS. 20C and 20D). Indeed, the precomplexed Fe(COMPOUND I).sub.3
drug is as potent as the parental COMPOUND I monomer in
cytotoxicity assays (FIG. 13A). Furthermore, Fe(COMPOUND I).sub.3
complex triggered apoptotic and DNA damage pathways, as measured by
c-PARP and .gamma.H2AX respectively, in MV-4-11 cells. Fe(COMPOUND
I).sub.3 also induced KLF4 and CDKN1A expression and inhibited MYC
in a dose-dependent manner (FIG. 13B). However, higher
concentrations of Fe(COMPOUND I).sub.3 were required to elicit an
equal response to parental COMPOUND I in the 24-hour assays (in
comparison with 5-day treatment in cytotoxicity assays) likely due
to a slower observed influx rate for precomplexed Fe(COMPOUND
I).sub.3 (FIG. 20E).
Example 15
COMPOUND I Stabilizes G-Quadruplex Sequences
[0197] The parental COMPOUND I and its intracellular Fe(COMPOUND
I).sub.3 form contain certain features, such as metal-coordinating
phenanthroline rings and planar structures, that may allow the
agent to function as a G-quadruplex (G4) DNA ligand. G4 is a
dynamic secondary DNA structure caused by guanine-rich regions
folding to form planar guanine tetrads, which stack on top of one
another. G4-specific sequences are found at telomeres and in the
promoters of many important oncogenes. G4 sequences serve as
regulators of gene expression and small-molecule ligands that
stabilize G4 quadruplexes have been exploited to downregulate
important oncogenes, such as KIT and MYC. Stabilization of G4
motifs in telomere DNA can cause inhibition of telomerase, telomere
instability, and deprotection, all of which can trigger DDR
pathways. Furthermore, origin of DNA replication sites overlap with
DNA G4 sequences, and stabilization of G-quadruplex structures at
such sites causes stalling of replication forks and cell-cycle
arrest.
[0198] The ability of COMPOUND I (parental monomeric form of the
drug) and Fe(COMPOUND I).sub.3 to bind and stabilize G4 sequences
using a modified FRET assay was evaluated (FIGS. 21 and 22).
TMPyP4, a well-known G4 ligand, and CX-5461, a clinical stage
molecule recently reported to have G4-binding properties, were
utilized as controls to assess the specificity of COMPOUND I and
Fe(COMPOUND I).sub.3 for G4-stabilizing activity. As expected,
CX-5461 was a potent stabilizer of all G4 sequences tested, and
TMPyP4 stabilized all G4 motifs except the G4 of the KIT gene
promoter (FIG. 13C). Interestingly, increasing concentrations of
Fe(COMPOUND I).sub.3 stabilized the G4 structures corresponding to
the MYC and KIT gene promoters, rRNA, and telomeres with a similar
potency to TMPyP4 (FIG. 13C; FIG. 22). Parental monomeric COMPOUND
I also showed time-dependent stabilization of G4 motifs, but it
demonstrated the greatest propensity for stabilizing the MYC G4
sequences (FIG. 13C).
[0199] To assess selectivity for G4 structures over nonspecific
interactions with ds-DNA, the FRET assay was repeated with a
self-complimentary oligo that forms a ds-DNA hairpin in solution.
Notably, Fe(COMPOUND I).sub.3 demonstrated a much higher degree of
selectivity for G4 structures over ds-DNA than did both CX-5461 and
TMPyP4, highlighting the fact that COMPOUND I is a more
discriminating G4 ligand (FIG. 13C; FIG. 21B). Gene expression
analyses showed that the expression of MYC and KIT was decreased in
AML cells in response to COMPOUND I treatment (RNA-seq data, MV4-11
cells 6-hour treatment), but levels of 45 s rRNA were not. The lack
of effect on 45 s rRNA may reflect differences in availability of
COMPOUND I and/or Fe(COMPOUND I).sub.3 into the rRNA-rich nucleolar
region of the nucleus. Nevertheless, COMPOUND I clearly can
stabilize G4 structures, which provides an explanation for the
inhibition of the expression of MYC and other genes. Without being
bound by any particular theory, it is hypothesized that
stabilization of G4 motifs by COMPOUND I results in single- and
double-strand breaks at replication forks and telomeres; this
G4-binding capacity of COMPOUND I identifies a mechanism by which
the drug triggers DDR pathways, cell-cycle arrest, and
apoptosis.
Example 16
Discussion
[0200] COMPOUND I is currently in clinical development for the
treatment of AML because of its efficacy in nonclinical models and
the fact that it did not produce myelosuppression in animals or in
its initial phase I trial in solid tumor patients. The data
reported here provide new insights into the mechanism of action of
this novel agent that point the way to more precise clinical
application and biomarker development. These studies confirmed that
COMPOUND I is a potent inducer of G.sub.0-G.sub.1 cell-cycle arrest
and apoptosis in AML cells. Additional new findings include that
COMPOUND I produces time- and concentration-dependent
downregulation of MYC through effects on both its promoters and
mRNA stability, that in many AML cell lines it induces the master
transcription factor and tumor suppressor KLF4, and that it induces
DNA damage. In addition, the pre-complexed iron form of COMPOUND I,
Fe(COMPOUND I).sub.3, causes comparable cytotoxic cellular effects,
including apoptosis, DNA damage, and downregulation of MYC
expression.
[0201] The discovery that COMPOUND I, whether in its parental
monomeric form or the Fe(COMPOUND I).sub.3 iron complex form,
stabilizes G4 motifs in DNA provides an explanation for many of the
pharmacodynamic effects of this drug. Stabilization of G4s is known
to disrupt telomere stability and stall replication forks,
resulting in single- and double-strand DNA breaks. Such
stabilization of G4 in the MYC promoter is thought to function as a
gene silencer. This, coupled with targeting of KIT and telomere G4
structures by COMPOUND I, provides a mechanism through which
COMPOUND I activates DDR pathways that coordinate cell-cycle arrest
and promote apoptosis in AML cells.
[0202] In addition, cells harboring BRCA1/2 mutations are
hypersensitive to COMPOUND I, further supporting a role for DNA
damage in COMPOUND I mechanism of action. COMPOUND I consistently
produced upregulation of CDKN1A, which mediates arrest in
G.sub.0-G.sub.1. In addition, CDKN1A can be induced after DNA
double-strand breaks to block cell-cycle progression to allow for
sufficient time to repair DNA. In combination with CDKN1A
induction, COMPOUND I increased KLF4 gene expression in many AML
cell lines, which is known to regulate CDKN1A as part of the
G.sub.1 cell-cycle checkpoint. KLF4 is also known to be upregulated
in response to DNA damage and plays a role in both G.sub.0-G.sub.1
arrest and apoptosis. The role of KLF4 in COMPOUND I mechanism of
action is of interest for future studies. Although the structure of
COMPOUND I suggests that it might be able to generate reactive
oxygen species, no such species have been detected using either
molecular sensors or changes in GSH in MV4-11, EOL1, or KG-1
cells.
[0203] Activation of CHEK1/2, stabilization of TP53, and induction
of E2F1 also indicate that the early events after COMPOUND I
treatment function to signal for cell-cycle arrest and DNA repair.
Cell-cycle arrest was detected by 2 hours after COMPOUND I
treatment, whereas upregulation of several proapoptotic factors at
both the RNA and protein levels was observed by 6 hours. In
addition to activating DNA repair processes, pCHEK1/2 and TP53 can
also play a role in triggering apoptosis. If DNA repair fails, p53
can activate apoptosis via upregulation of BAX, BAD, BBC3, or
PMAIP1. Increased expression of these proapoptotic factors was
detected by RNA-seq analysis of COMPOUND I-treated MV4-11 cells. It
is known that caspase-dependent cleavage of PARP1 is required for
apoptosis to proceed. COMPOUND I produced robust and early PARP1
cleavage, adding further credence to the hypothesis that COMPOUND I
functions by triggering DDR pathways. This suggests a level of DNA
damage that is catastrophic to the cell and an alteration of
transcriptional programs that skew the cell toward apoptosis. MYC
dysregulation is a common oncogenic driver in multiple
malignancies, which makes it an attractive potential therapeutic
target. However, targeting MYC is challenging due to the complexity
of MYC regulation and signaling. Recently, repression of MYC
expression by BET bromodomain inhibitors has proven effective at
triggering apoptosis in leukemia cells. However, bromodomain
proteins are present on all active genes, and inhibition of
bromodomain proteins can cause severe toxicities and
myelosuppression. COMPOUND I produced a decrease in MYC expression
at both the RNA and protein levels in all AML cell lines tested,
and downregulation of MYC paralleled its cytotoxic potency in
different AML cells. Higher MYC levels in AML lines than in PBMCs
from healthy donors were detected, which may be linked to the
differential effect of COMPOUND I on these types of cells. Recent
work demonstrated that coordinated upregulation of TP53 and
downregulation of MYC led to efficient clearing of leukemic stem
cell populations in CML. COMPOUND I treatment of MV4-11 produced
this same effect, which provides an additional rationale for its
development. It has been reported that higher MYC expression
correlates with a poor clinical outcome in epithelial ovarian
cancer and neuroblastoma, suggesting that COMPOUND I may have a
beneficial effect against these malignancies. Collectively, this
data demonstrate a multifaceted mechanism of action for COMPOUND I,
primarily through engagement of G-quadruplex structures, that is
uniquely suited to targeting hematopoietic malignancies. Moreover,
COMPOUND I represents a first-in-class MYC inhibitor that does not
cause myelosuppression, making it particularly appropriate for the
management of AML patients with compromised bone marrow
function.
[0204] The foregoing examples and description of certain
embodiments should be taken as illustrating, rather than as
limiting the present invention as defined by the claims. As will be
readily appreciated, numerous variations and combinations of the
features set forth above can be utilized without departing from the
present invention as set forth in the claims. All such variations
are intended to be included within the scope of the present
invention. All references cited are incorporated herein by
reference in their entireties.
[0205] It is to be understood that, if any prior art publication is
referred to herein, such reference does not constitute an admission
that the publication forms a part of the common general knowledge
in the art in any country.
[0206] The disclosures of all publications, patents, patent
applications and published patent applications referred to herein
by an identifying citation are hereby incorporated herein by
reference in their entirety.
Sequence CWU 1
1
15119DNAArtificial SequenceABCG2 oligonucleotide primer 1ttaggattga
agccaaagg 19221DNAArtificial SequenceABCG2 oligonucleotide primer
2taggcaattg tgaggaaaat a 21320DNAArtificial SequenceMYC
oligonucleotide primer 3gagcagcagc gaaagggaga 20420DNAArtificial
SequenceMYC oligonucleotide primer 4cagccgagca ctctagctct
20519DNAArtificial SequenceMYC oligonucleotide primer 5ccgcatccac
gaaactttg 19623DNAArtificial SequenceMYC oligonucleotide primer
6gggtgttgta agttccagtg caa 23727DNAArtificial Sequence28s RNA
oligonucleotide primer 7agtagcaaat attcaaacga gaacttt
27821DNAArtificial Sequence28s RNA oligonucleotide primer
8acccatgttc aactgctgtt c 21920DNAArtificial SequenceMYC
oligonucleotide primer 9cagtagaaat acggctgcac 201020DNAArtificial
SequenceMYC oligonucleotide primer 10ttcgggtagt ggaaaaccag
201133DNAArtificial SequenceG-quadruplex Telomere
oligonucleotidemisc_feature(1)..(1)FAM (fluorescein) fluorescent
dye covalently attachedmisc_feature(33)..(33)BHQ1 (Black Hole
Quencher 1) non-fluorescent chromophore covalently attached
11gggttagggt tagggttagg gttagggtta ggg 331229DNAArtificial
SequenceG-quadruplex MYC oligonucleotidemisc_feature(1)..(1)FAM
(fluorescein) fluorescent dye covalently
attachedmisc_feature(29)..(29)BHQ1 (Black Hole Quencher 1)
non-fluorescent chromophore covalently attached 12ccatggggag
ggtggagggt ggggaaggt 291333DNAArtificial SequenceG-quadruplex KIT
oligonucleotidemisc_feature(1)..(1)FAM (fluorescein) fluorescent
dye covalently attachedmisc_feature(33)..(33)BHQ1 (Black Hole
Quencher 1) non-fluorescent chromophore covalently attached
13ttatagggag ggcgctggga ggagggagga gac 331433DNAArtificial
SequenceG-quadruplex rRNA oligonucleotidemisc_feature(1)..(1)FAM
(fluorescein) fluorescent dye covalently
attachedmisc_feature(33)..(33)BHQ1 (Black Hole Quencher 1)
non-fluorescent chromophore covalently attached 14aataagggtg
gcggggggta gaggggggta ata 331520DNAArtificial SequenceG-quadruplex
ds_DNA oligonucleotidemisc_feature(1)..(1)FAM (fluorescein)
fluorescent dye covalently attachedmisc_feature(10)..(11)linked by
Spacer 18 which is a hexaethylene glycol chain that is 18 atoms
longmisc_feature(20)..(20)BHQ1 (Black Hole Quencher 1)
non-fluorescent chromophore covalently attached 15tatagctata
tatagctata 20
* * * * *
References